**4. Brain imaging evidence**

Recent advances in neuroimaging techniques make it possible to identify the brain-based factors that facilitate successful reading outcomes. Importantly, brain imaging may provide innovative solutions to improve education curriculums and lead to improvements in reading results in young children.

Over the last decades, neuroimaging studies focused on identifying brain markers that are the cause of dyslexia (see reviews: [48, 49]). Although researchers are far from concluding that the brain markers causing dyslexia, we have learned about the neural basis of reading acquisition. For instance, a left-lateralized brain network, including temporoparietal and occipitotemporal cortices, is critical to facilitate skilled reading [50, 51] (see **Figure 1**). High white matter integrity in accurate fasciculus (AF) predicts better reading outcomes in children at risk for dyslexia [52]. AF is a tract connecting Broca's area and Wernicke's area, related to reading ability [53–55] (see **Figure 1**). If neuroimaging measures can identify children at risk for reading difficulties before they even start to learn to read in school, early emergent reading interventions can be applied to help them overcome the risk of developing reading difficulties in school years. Only a limited number of studies have specifically investigated the relationship between emergent reading environments and neuroimaging data.

Hutton et al. used StimQ-P questionnaire [56] to quantify the cognitive simulation at home and identified that functional magnetic resonance imaging (fMRI) data during a storying comprehension task presented stronger activity for those children with higher StimQ-P Reading scores [57]. They reported that higher StimQ Reading scores were associated with stronger activation in occipital cortices, including lateral occipital gyrus and precuneus, which can be attributed to mental imagery evoked during story listening [58]. Their study sample includes nineteen 3- to 5-year-old children from a longitudinal study of healthy brain development. In preschool children listening to stories, greater home reading exposure was positively related to activation of left posterior occipital fusiform, lateral occipital, posterior inferior temporal, posterior middle temporal, posterior cingulate, and angular gyri and left precuneus (household income is controlled). Their finding suggests that

#### **Figure 1.**

*Brain regions and white matter tracts related to reading on a 3D rendered brain. Red: accurate fasciculus (AF), green: superior longitudinal fasciculus (SLF).*

brain-based markers exist as a result of parent-child reading in early childhood. Thus, emergent reading shall be promoted and may help shape the developing brain and better prepare a child for formal reading instructions in school.

Developmental dyslexia (DD) has strong genetic basis [59], and family history of DD can increase a child's chance to develop reading difficulties by 34–56% [60–62]. In order to identify children at risk for DD, familial risk can be used as a good indicator. One group led by Dr. Nadine Gaab in Boston Children's Hospital has done pioneer work in this research field [48, 52, 63–70]. For the first time, they examined the relationship between home literacy environment (HLE) and the neural basis of phonological processing in beginning readers with family history of DD (n = 29, first-degree relative who has reading difficulties) and without family history of DD (n = 21) [67]. This study aimed to identify brain mechanism of how HLE affects reading development in beginning readers. SES was controlled in this study in order to isolate the effects only by HLE. In reading-related brain regions (e.g., left inferior/ middle frontal and right fusiform gyri), stronger correlations between HLE composite scores and brain activations were present in children without familial risk than those with familial risk. In the nonreading-related brain region (e.g., right precentral gyrus), stronger correlations existed in children with familial risk than those without familial risk. These findings suggest that genetic predisposition for DD alters contributions of HLE to brain activation. Specifically, typically developing children can benefit more from better HLE than children with familial risk for DD. Therefore, enhanced HLE is especially important for children with familial risk for DD to have the same impact as for typically developing children.

Shared parent-child reading is one of the important factors in emergent reading. A recent study demonstrated increased activation and functional connectivity in children who are more deeply engaged during shared reading in 22 motherdaughter pairs [71]. The same group also associated shared reading quality scores with brain activation, and they found a positive correlation between shared reading quality scores with activation in left-hemispheric regions supporting expressive and complex language, social-emotional integration, and working memory in 22 healthy, 4-year-old girls from low SES [72]. Their findings suggest that the use of shared parent-child reading is crucial for emergent reading experience, but the quality of this experience has also a strong impact on brain development. Especially for those at-risk families, improvements of the quality of shared reading can promote healthy brain development and better prepare a child for future success in school.

Morken et al. [73] used a longitudinal study design to examine the differences of cortical connectivity in the brain during reading tasks between children with dyslexia and children with typical reading development through dynamic causal modeling (DCM) [74]. They included five regions (inferior frontal gyrus, precentral gyrus, superior temporal gyrus, inferior parietal lobule, and occipitotemporal cortex) in their effective brain connectivity model [74]. They found that effective connectivity between the inferior frontal gyrus and the occipitotemporal cortex during reading tasks changes during reading acquisition. In addition, the group readers with dyslexia presented different developmental trajectory than the control group. The control group actually seemed to downregulate or stabilize connection strength over time, whereas the dyslexia group started out at a level well below the control group, followed by an increase in connectivity from 6 to 8 years and then a downregulation from 8 to 12 years. The general downregulation of connectivity in the control group might reflect that they need these connections to establish reading skills initially, and then, the connections are no longer needed after later automaticity is established. The dyslexia group showed late development of some connections in occipitotemporal cortices. However, they seem to show overcompensation

**229**

*Emergent Reading and Brain Development DOI: http://dx.doi.org/10.5772/intechopen.82423*

develop simultaneously.

**5. Discussion**

around age 8, followed by normalization before age 12. Importantly, the dyslexia group was clearly lagging behind in the development of the brain networks at the age of 8 (emergent reading stage), suggesting emergent reading stage is critical. Younger et al. also used a longitudinal study design and found decreases in connectivity for most connections from the first (T1) to the second (T2) time point about 2–3 years apart, regardless of changes in reading skill in 59 typical developing children [75]. But they found a significant decrease in the dorsal, decoding processing pathway from fusiform gyrus (FG) to inferior parietal lobule (IPL) for the group who improved more from the first to the second time point, suggesting that the improvements in reading skills lead to a decreased reliance on the dorsal pathway (decoding processing pathway) in the brain. The high and low improving groups did not differ in behavioral performance at T1, and high improvers showed greater connectivity between FG and IPL at T1 compared to the low improvers. The dorsal pathway facilitates phonological processing, which is necessary for development of the ventral pathway supporting automatic processing of visual word forms. However, there is no sequential relationship between the two routes. They may

Yu et al. studied 28 children over three stages (pre-reading, beginning reading, and emergent reading) and found decreases in neural activation in the left inferior parietal cortex (LIPC) during an audiovisual phonological processing task [69]. Seed-based brain network analysis revealed increases in connection strength in the brain network of children with above-average gains in phonological processing but decreases in connection strength in the brain network of children with below-average gains in phonological processing measured by Comprehensive Test of Phonological Processing (CTOPP). Moreover, the connection strength between LIPC and the left posterior occipitotemporal cortex (LpOTC, BA 18) at the prereading stage significantly predicted reading skills at the emergent reading stage.

This chapter demonstrates the view of emergent reading and brain imaging evidence supporting advocacy for emergent reading. Emergent reading emphasizes the developmental continuum aspect of learning to read and the importance of

Both behavioral and imaging studies on DD suggest that early reading skills are essential to the later development of reading. Most children start formal reading curriculum in kindergarten; however, at that time, many factors (genetic, SES, HLE, etc.) have already affected the future reading development. Moreover, early

Brain regions (left inferior/middle frontal gyrus, bilateral fusiform gyri, and right anterior superior temporal gyrus) were identified to be especially sensitive to differences of early language/literacy exposure in beginning readers [67]. A richer HLE corresponded to increased brain activations during a phonological processing task [67] and increased brain activations related to high reading proficiency [76] demonstrated the underlying neural basis of reading. Among the children with a familial risk for DD, only around 50% of them will develop DD. The imaging evidence implies that a rich HLE might be one of the protective factors in reading development especially for children with a familial risk for DD. Future longitudinal studies are needed to examine how HLE contributes to the development of reading

Advocating emergent reading can benefit all children who are learning to read and especially those who are also at risk for DD. It is clear that aspects of HLE

networks in the brain and its role as a protective factor in general.

reading-related behaviors occurring before school.

interventions work more effectively.

#### *Emergent Reading and Brain Development DOI: http://dx.doi.org/10.5772/intechopen.82423*

*Early Childhood Education*

brain-based markers exist as a result of parent-child reading in early childhood. Thus, emergent reading shall be promoted and may help shape the developing brain

Developmental dyslexia (DD) has strong genetic basis [59], and family history of DD can increase a child's chance to develop reading difficulties by 34–56% [60–62]. In order to identify children at risk for DD, familial risk can be used as a good indicator. One group led by Dr. Nadine Gaab in Boston Children's Hospital has done pioneer work in this research field [48, 52, 63–70]. For the first time, they examined the relationship between home literacy environment (HLE) and the neural basis of phonological processing in beginning readers with family history of DD (n = 29, first-degree relative who has reading difficulties) and without family history of DD (n = 21) [67]. This study aimed to identify brain mechanism of how HLE affects reading development in beginning readers. SES was controlled in this study in order to isolate the effects only by HLE. In reading-related brain regions (e.g., left inferior/ middle frontal and right fusiform gyri), stronger correlations between HLE composite scores and brain activations were present in children without familial risk than those with familial risk. In the nonreading-related brain region (e.g., right precentral gyrus), stronger correlations existed in children with familial risk than those without familial risk. These findings suggest that genetic predisposition for DD alters contributions of HLE to brain activation. Specifically, typically developing children can benefit more from better HLE than children with familial risk for DD. Therefore, enhanced HLE is especially important for children with familial risk for DD to have

Shared parent-child reading is one of the important factors in emergent reading. A recent study demonstrated increased activation and functional connectivity in children who are more deeply engaged during shared reading in 22 motherdaughter pairs [71]. The same group also associated shared reading quality scores with brain activation, and they found a positive correlation between shared reading quality scores with activation in left-hemispheric regions supporting expressive and complex language, social-emotional integration, and working memory in 22 healthy, 4-year-old girls from low SES [72]. Their findings suggest that the use of shared parent-child reading is crucial for emergent reading experience, but the quality of this experience has also a strong impact on brain development. Especially for those at-risk families, improvements of the quality of shared reading can promote healthy brain development and better prepare a child for future success in

Morken et al. [73] used a longitudinal study design to examine the differences of cortical connectivity in the brain during reading tasks between children with dyslexia and children with typical reading development through dynamic causal modeling (DCM) [74]. They included five regions (inferior frontal gyrus, precentral gyrus, superior temporal gyrus, inferior parietal lobule, and occipitotemporal cortex) in their effective brain connectivity model [74]. They found that effective connectivity between the inferior frontal gyrus and the occipitotemporal cortex during reading tasks changes during reading acquisition. In addition, the group readers with dyslexia presented different developmental trajectory than the control group. The control group actually seemed to downregulate or stabilize connection strength over time, whereas the dyslexia group started out at a level well below the control group, followed by an increase in connectivity from 6 to 8 years and then a downregulation from 8 to 12 years. The general downregulation of connectivity in the control group might reflect that they need these connections to establish reading skills initially, and then, the connections are no longer needed after later automaticity is established. The dyslexia group showed late development of some connections in occipitotemporal cortices. However, they seem to show overcompensation

and better prepare a child for formal reading instructions in school.

the same impact as for typically developing children.

**228**

school.

around age 8, followed by normalization before age 12. Importantly, the dyslexia group was clearly lagging behind in the development of the brain networks at the age of 8 (emergent reading stage), suggesting emergent reading stage is critical.

Younger et al. also used a longitudinal study design and found decreases in connectivity for most connections from the first (T1) to the second (T2) time point about 2–3 years apart, regardless of changes in reading skill in 59 typical developing children [75]. But they found a significant decrease in the dorsal, decoding processing pathway from fusiform gyrus (FG) to inferior parietal lobule (IPL) for the group who improved more from the first to the second time point, suggesting that the improvements in reading skills lead to a decreased reliance on the dorsal pathway (decoding processing pathway) in the brain. The high and low improving groups did not differ in behavioral performance at T1, and high improvers showed greater connectivity between FG and IPL at T1 compared to the low improvers. The dorsal pathway facilitates phonological processing, which is necessary for development of the ventral pathway supporting automatic processing of visual word forms. However, there is no sequential relationship between the two routes. They may develop simultaneously.

Yu et al. studied 28 children over three stages (pre-reading, beginning reading, and emergent reading) and found decreases in neural activation in the left inferior parietal cortex (LIPC) during an audiovisual phonological processing task [69]. Seed-based brain network analysis revealed increases in connection strength in the brain network of children with above-average gains in phonological processing but decreases in connection strength in the brain network of children with below-average gains in phonological processing measured by Comprehensive Test of Phonological Processing (CTOPP). Moreover, the connection strength between LIPC and the left posterior occipitotemporal cortex (LpOTC, BA 18) at the prereading stage significantly predicted reading skills at the emergent reading stage.

## **5. Discussion**

This chapter demonstrates the view of emergent reading and brain imaging evidence supporting advocacy for emergent reading. Emergent reading emphasizes the developmental continuum aspect of learning to read and the importance of reading-related behaviors occurring before school.

Both behavioral and imaging studies on DD suggest that early reading skills are essential to the later development of reading. Most children start formal reading curriculum in kindergarten; however, at that time, many factors (genetic, SES, HLE, etc.) have already affected the future reading development. Moreover, early interventions work more effectively.

Brain regions (left inferior/middle frontal gyrus, bilateral fusiform gyri, and right anterior superior temporal gyrus) were identified to be especially sensitive to differences of early language/literacy exposure in beginning readers [67]. A richer HLE corresponded to increased brain activations during a phonological processing task [67] and increased brain activations related to high reading proficiency [76] demonstrated the underlying neural basis of reading. Among the children with a familial risk for DD, only around 50% of them will develop DD. The imaging evidence implies that a rich HLE might be one of the protective factors in reading development especially for children with a familial risk for DD. Future longitudinal studies are needed to examine how HLE contributes to the development of reading networks in the brain and its role as a protective factor in general.

Advocating emergent reading can benefit all children who are learning to read and especially those who are also at risk for DD. It is clear that aspects of HLE

(e.g., shared reading) before a child entering kindergarten or preschool benefit the later reading development.


2.4.2. Acute and Chronic Tests on *D. magna*

The acute toxicity test on *D. magna* for both ELT-dg and ELT-dp suspensions was conducted in a 48 h exposure under static conditions to 100.0 mg/L [48]. In particular, we evaluated the immobilization, defined as organisms not able to swim within 15 s after gentle agitation. Specimens (daphnid age < 24 h) obtained by ChemService Controlli e Ricerche s.r.l. breeding were exposed in 40 mL beakers with pre-aerated (oxygenation > 5.62 mg/L) reconstituted water (see Supplementary Materials for composition), with 4 replicates *per* treatment (5 daphnids *per* beaker) at 20 ◦C, with 16 h of light and 8 h of darkness (1188–1401 lx). The immobilization was checked after 24 and 48 h. The chronic test on *D. magna* for both ELT-dg and ELT-dp was conducted in 21 days of exposure in semi-static condition (renewal 3 times *per* week) to 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L [50]. In this context, after 21 days of exposure, we evaluated the mortality rate and reproduction parameters (total number of living offspring). Specimens (daphnid age < 24 h) were exposed in 50 mL beakers with pre-aerated (oxygenation > 5.00 mg/L) reconstituted water (see the Supplementary Materials for the composition) with 10 replicates *per* treatment (10 daphnids *per* treatment, 1 specimen *per* beaker) at 20 ◦C, with 16 h of light and 8 h of darkness (1236–1467 lx). During exposure, daphnids were feed with *P. subcapitata* after the water change.


*3.6. Morphology*

The morphology of a photocatalyst also affects the performance, for the surface area and the surface active sites are influenced by the structure. Photocatalysts have different morphologies, including 3D and porous morphologies, nanosheets, nanorods, nanoflowers, and nanowires [14,19,25,32,34,47,77,103]. Recently, Amirav et al. studied tunable nanorod heterostructures. They demonstrated that a longer CdSe seeded rod was more active than a shorter rod of the same diameter, as the surface active sites were located further apart. However, nanorods with comparable rod lengths but smaller diameters will provide higher activity [102]. Panmand et al. reported that more structural defects and surface states are created on CdS decorated Bi2S3 nanowires. Figure 7 presents the morphology of a composite photocatalyst. The photogenerated charge carriers of photocatalysts with such defect energy levels can be effectively separated, leading to enhanced photocatalytic activity [104].

**Figure 7.** (**a**) HRTEM image; (**b**) SAED pattern of CdS decorated Bi2S3 nanowires; magnified HRTEM images of (**c**) Bi2S3 nanowire; (**d**) CdS nanoparticle [104].

In addition, a 2D morphology (such as a nanosheet) can help to improve the photocatalytic activity. Zhang et al. reported that ZnIn2S4/g-C3N4 heterojunction nanosheets demonstrated higher H2

production rates compared to single heterojunction components [101]. The contact of the components to form heterojunctions is very important [105]. A nanorod array structure with a small inter-rod distance will not easily form heterojunctions with close contact between different components. In contrast, a 2D heterostructure has enhanced photocatalytic performance because the van der Waals interaction of the 2D heterostructure junction between 2D metals (1T-MoS2) and the 2D semiconductor (O-g-C3N4) minimizes the Schottky barrier, thus improving the efficiency of charge transfer [106].

Moreover, the notable high hydrogen production rate and good stability achieved by mesoporous monoclinic CaIn2S4 with surface nanostructures implies the importance of structure in enhancing the H2 evolution rate. Ding et al. reported that monoclinic CaIn2S4 (m-CaIn2S4) exhibits a lower ability than cubic CaIn2S4 (c-CaIn2S4) to absorb visible light, but it also has better photocatalytic performance. The better performance results from the larger surface area, higher pore volume, more negative conduction band potential, and efficient separation of photoexcited carriers of m-CaIn2S4 [107].

Furthermore, a core–shell structure will also benefit the photocatalyst, as it may have an increased surface area and change the surface properties. For example, Chang et al. reported that the growth of Ag2S-ZnO@ZnS core–shell nanorods on metal wire mesh had modified the surface from hydrophobic to superhydrophilic. In addition, the H2 production activity also increased with the increasing thickness of the ZnS shell [32]. A similar trend was also evidenced in studies of NiCo2O4@ZnS and Fe3O4@ZnS core shell photocatalysts [72]. Table 11 presents the performances of photocatalysts with different structures.

**Table 11.** Photocatalytic activity of photocatalysts with different structures.

#### 3.6.1. Facet Effect

It has been reported that the photocatalysis reaction mainly occurs at the surface of the photocatalyst. The exposure of certain facets leads to greatly improved activity of the photocatalysts, known as the facet effect. Therefore, the preparation of photocatalysts with specific morphologies and structures is an important topic in the photocatalysis field. The facet effect has been observed for some oxide-based photocatalysts. Li et al. [109] reported that efficient separation of photoexcited electron–hole pairs can occur between different facets of photocatalytic nanomaterials. In comparison with their analogs with randomly distributed cocatalysts, selective deposition of a reduction cocatalyst and an oxidation cocatalyst on the {010} and {110} facets of BiVO4 leads to higher photocatalytic activity. Ohno et al. [110] found the effect of facets on the photocatalytic activity of TiO2 photocatalysts. For the rutile TiO2 nanomaterials, the {110} facet can act as an effective reduction site, and the {011} facet can offer a site for effective oxidation. TiO2 photocatalysts show high activity because of the synergistic effect between the {110} and {011} facets.

Similar results were also found for the sulfide-based photocatalysts. Song et al. found that, in comparison with 2-D Cu2MoS4 nanosheet with the exposed {001} facet, Cu2MoS4 nanotube with the exposed {010} facet exhibited effectively improved the performance for photocatalytic degradation and water splitting [111]. Shen et al. reported that the crystal facets of ZnIn2S4 with a 3D-hierarchical persimmon-like structure will influence the photocatalytic activity of ZnIn2S4. Extending the reaction time did not reveal any significant influences on the band gap or surface area of ZnIn2S4. Hence, the increase in the percentage of the {006} facet enhances hydrogen production [108]. The atomic structure of the {006} facet mainly consists of unsaturated metal cations. During the H2 generation reaction, the exposed unsaturated Zn and In cations of ZnIn2S4 will attract S2<sup>−</sup> and SO3 − anions, which may help the oxidation process and speed up the consumption of photogenerated holes (Figure 8). This impedes the electron–hole recombination process, leading to improved photocatalytic activity.

**Figure 8.** Schematic illustration of the hydrogen generation reaction on the {006} facets of the Pt loaded ZnIn2S4 photocatalysts. Figure adapted from reference [108].

#### 3.6.2. Light Trapping (Light Harvesting)

It is reported that properly patterned surface textures can lead to dramatically enhanced light absorption by the photocatalyst because of the light trapping effect [32]. Some textured structures including nanowire arrays [112], ordered mesoporous structures [113], micro-hole arrays [114], and hemisphere-array films [115,116]—are able to increase the light harvesting and photocatalytic performance of photocatalysts. Zhang et al. [112] reported that a 3D ZnO nanowire array–CdS sample exhibited substantial light-trapping enhancement in the visible light region. A schematic illustration of interface scattering when the light was irradiated on the surface of porous photocatalyst is provided in Figure 9 [116]. To enhance the light absorption efficiency, these surface textures allow multiple reflections and light scattering within the nanostructures. The incident light can travel through the cavities and decrease the optical loss. Efficient light trapping can be achieved by tuning the shape and roughness of the textured surface [117].

**Figure 9.** Schematic illustration of interface scattering due to nanograss decorated pore-array photocatalysts. Figure adapted from [116].


*Microfluidics for Time-Resolved Small-Angle X-Ray Scattering DOI: http://dx.doi.org/10.5772/intechopen.95059*

compression, or focusing, of the central flow, and decreases the mixing times significantly by reducing the diffusion length. There are two main categories for microfluidic devices with hydrodynamic focusing: coaxial tube and planar on-chip devices. The design can be defined depending on the use, with adapted geometries for fast mixing (**Figure 2A**), gradients (**Figure 2B**), specific nanoparticle growth reactions or self-assembly processes (**Figure 2C**) [15].

The simplest type of coaxial tube reactors is a device consisting of two concentric capillaries (**Figure 2D**), which are connected to a channel where a central flow is injected through the inner capillary, with sheath flow injected from the outer layer. Coaxial tube microreactors find various applications, but are typically used as the interface for droplet-based reactors, as the transition from flow to droplet generation, dripping and jetting is defined by the flowrate of the outer sheath flow. However, this approach is of limited utility in time resolved mixing applications, as it only offers limited mixing geometries. Further limitations abound in the fabrication process for coaxial designs, which requires multiple steps and precise alignment and assembly of the parts [15].

On-chip hydrodynamic focusing devices can be differentiated in twodimensional (2D) and three-dimensional (3D) devices. In two-dimensional hydrodynamic focusing devices, the central flow is focused in the horizontal plane. The simplest geometry for a 2D device is a Y- or T-shaped mixer (**Figure 2E**), where the cross-sectional diffusion is broadened at the channel walls in comparison to the centre. However, this design is highly limited regarding flow stability and focusing and susceptible to variation in these parameters in operation. To avoid this, cross-

#### **Figure 2.**

This understanding of this pressure-driven, steady-state flow in microfluidic

microfluidics, the channel cross-sections can be of various shapes, depending on the application and fabrication method. Eqs. (13) to (16) describe the velocity field and hydraulic resistance for spherical and rectangular cross-sections, which are the most common geometries used for the devices described in this chapter. The derivation of those values is exceedingly more complicated for arbitrary channel cross-section

The first design consideration is how the device will enable time-resolved measurement of phenomenon. This can be achieved in two different ways. The first, and conceptually simplest method, is a static experiment, where a sample is firstly mixed and then introduced into a monitoring chamber and measured repeatedly at defined time periods. The most common apparatus for this style of measurement this is a *stopped flow* device, where mixing is achieved rapidly, and then flow is stopped as soon as the homogenously mixed sample fills the monitoring chamber. The measurement is triggered as soon as the flow is stopped, and generally continues as rapidly as possible until the reaction reaches completion. The second method is to use a continuous flow system, where the mixed sample is introduced into a flow-through system, and temporal measurements are achieved by varying

the distance between the mixing point and the sampling point.

Both styles of devices have advantages and disadvantages, and the choice depends strongly on several experimental considerations, including the time domain of the reaction, mixing efficiency, sample volume constraints, and sample chemistry constraints (e.g. resistance to photobleaching, or radiation damage). Stopped flow measurements are favoured when there is a small volume of sample that is resistant to measurement induced damage (for example a flurophore that is resistant to photobleaching), where the reaction is not extremely fast, and where the experimental measurements are not slow. In stopped flow measurements the initial point in the measurement is always some degree of time post the start of the reaction (given the time it takes to fill the sample cell, stop the flow and take the first measurement), and the temporal resolution of the measurement is given by the speed at which the measurement can be taken. However, agglomeration of the reacting sample on the channel walls can influence the quality of measurements and, due to the ongoing reaction, leads to only a small window that can be detected before the experiment needs to be repeated. Alternatively, continuous flow measurements favour samples that are sensitive to the measurement, are very rapid, and require temporal resolution finer then the measurement speed of the instrument. Continuous flow measurements allow for measurement very close to the point of mixing, temporal resolution is given by the spatial resolution of the measurement, and the time taken to travel to the point of measurement. Further, the deadtime and temporal resolution is heavily influenced by flowrate, allowing for fine control across many temporal regions. As a result, the observation of the reaction can be precisely controlled. It needs to be considered that continuous flow measurements need more sample volume in comparison to stopped flow methods,

The basis of hydrodynamic focusing lies in a central solution that flows with a lower flowrate within an outer sheath fluid with a higher flowrate. This enables the

channels is the basis of liquid handling in lab-on-chip systems. Especially in

shapes.

*2.1.2 Continuous flow vs. stopped flow*

*Advances in Microfluidics and Nanofluids*

to provide a constant flow profile.

*2.1.3 Hydrodynamic focusing*

**18**

*Left: Scheme of microfluidic features for kinetic investigations in flow in a cross shaped mixer. (A) Hydrodynamically focused Centre stream for fast mixing experiments. (B) Mapping of concentration gradient across and along the channel through interdiffusion of different liquids from main and side channels. (C) Nucleation and growth of nanoparticles or self-assembly processes of nanomaterials as a function of time along the outlet channel. Schematic comparison of the provided time scales in continuous and stopped flow microfluidic devices. Right: Schematic illustration of microfluidic devices with various channel cross designs with the corresponding cross-sections through the outlet channel. (D) Coaxial tube reactor with two concentric channels/capillaries. (E) Y-shaped design, where mixing is solely based on diffusion. (F) Cross-shaped geometry at the inlets for hydrodynamic focusing. (G) Two-cross-section geometry, also known as double-focus device, where three different solutions can be introduced into the channels. Solutions introduced into the first sidechannel (SC1) act as an inert buffer between reactants in main channel (MC) and second side-channel (SC2). (H) and (J) multilayer designs of the geometries from (F) and (G), respectively, avoiding contact between the central stream and channel walls. (K) Hybrid device consisting of multilayer focusing device (J) and an inserted glass capillary as outlet channel.*

shaped geometries can be employed, where the central flow is focused from both incoming side channels (**Figure 2F**). This provides good control over the thickness of the central stream. Furthermore, it allows a well-defined sample composition that can be adapted by variation of the volume flow in each inlet individually. To adapt the device design to multi-step synthesis, several side channels can be added to introduce additional reactants (**Figure 2G**). Thus planar on-chip hydrodynamic focusing is more highly favoured for flexible mixing devices, and is used much more often in general microfluidic designs.

**Lithography.** One of the most powerful methods in microfabrication is lithography. It can be differentiated by the type of radiation used, e.g. photolithography, electron-beam lithography, or X-ray lithography. With these different lithography methods, structures with sizes between 0.2 and 500 μm in hard materials like glass, or between 0.5 and 500 μm in soft materials like polymers, can be achieved. In the most common form of lithographic fabrication, a UV blocking mask is generated from the CAD model, and adhered to a silicon wafer. The master model for fabrication is then generated by photolithography, where the masked wafer is coated in a photosensitive epoxy monomer solution, and UV-cured. In general these silicon masters are then used to generate working devices via soft lithographic replication.

The most common approach is to use the master chip as a mould, and poly

which part of the structure was UV-cured on the silicon master.

*Microfluidics for Time-Resolved Small-Angle X-Ray Scattering*

*DOI: http://dx.doi.org/10.5772/intechopen.95059*

time [19, 20].

wider channels [21, 22].

*2.2.2 Device materials*

**21**

(dimethylsiloxane) (PDMS) to form an imprinted device, which can be bonded to a glass-slide or a second PDMS part to form the final microfluidic device [16–18]. The replicated structure can be a positive or negative of the initial design, depending on

**Hot embossing and micromoulding.** Hot embossing is a micromoulding technique that uses thermoplastic polymers to imprint structures at elevated temperatures. It usually uses high-temperature polymers, e.g. PMMA, PC, PI, PE, PVC or PEEK, which are heated above their glass transition temperature (*Tg*) before being pressed into a mould with high pressure. The moulds have to withstand the applied pressure and high temperatures, and are often made of metal or silicon, fabricated via etching, lithography in combination with electroforming and moulding or CNC (Computerised Numerical Control)-machining. The accuracy of hot embossing is in the order of tens of nanometres, making it possible to obtain high aspect ratios of structure, while being a low cost and easy procedure. It is often used with a defined and tested device design as high throughput method with a very short fabrication

**3D printing.** Within the last decade, 3D printing technologies have advanced to

Based on the desired purpose of the microfluidic chip, device materials must fulfil specific criteria. The most important requirements which will be addressed in this chapter are solvent stability, ease of fabrication, and optical and X-ray transparency. **Solvent stability.** Microfluidics deals with the manipulation of liquids, which means that the device material has to be resistant and inert to the solvent. This becomes especially relevant when using organic solvents, as they often cause swelling or dissolution of standard polymeric device materials. Swelling leads to deformation, which can cause channel closure. A number of device materials have been tested with regard to resistance to some common solvents for nanoparticle synthesis and self-assembly processes (**Table 1**). It is clear from these results that careful

**Ease of fabrication.** While the solvent is important, it is also essential to con-

sider the difficulty of working with the various polymers, and the end

stereolithography (SLA) or selective laser sintering (SLS), have been developed for various materials like polymers, resins, ceramics or metals. It is possible with these techniques to produce a complete microfluidic device in one step. The device material and process can thereby be selected with regard to required mechanical and chemical properties of the device. A channel size resolution of few hundred micrometres can be achieved, making this approach preferred for devices with

astonishing precision, in size-regimes down to the micrometre scale. Additive

manufacturing technologies like fused deposition modelling (FDM),

selection of polymer is necessary for long term stability.

However, there are further considerations that need to be taken into account, particularly for chemical reactions or self-assembly processes. In coaxial and 2D channel geometries the interface of reacting solutions is in contact with the channel walls, and particles or macromolecules can stick and agglomerate on the channel surface and disturb the laminar flow conditions. Furthermore, this accumulation interferes with analytical investigations and, in the worst case, can cause complete blockage of the channel.

To avoid channel contact, three-dimensional channel geometries can be used. 3D hydrodynamic focusing requires both horizontal and vertical focusing of the central reactant stream, leading to a complete enclosure with liquid from all sides (**Figure 2H** and **J**). The device design can be optimised to reach homogeneous mixing without integrating specific mixing regions before the measurement part of the microfluidic chip. Additionally, these devices are simple to fabricate and have easily adjustable designs. The most common design to achieve 3D hydrodynamic focusing is through multi-layer on-chip devices, which require precise alignment as part of the fabrication process. Alternate methods are single layer devices or novel fluid manipulation technologies like "microfluidic drifting", which introduces lateral drifts or counter-rotating vortex forces to achieve vertical and horizontal flow focusing. These alternatives require less alignment in manufacturing and are thus much more user friendly in regards to fabrication.

All previously described device geometries can be used to produce droplets, liquid jets and sprays under the right flow conditions, including ultra-high flowrates. These devices are not limited to constrained flows inside channels, and for time-dependent studies the use of free liquid jets is preferred. Measurements of free jets have significant advantages in many optical measurements, as there is little to no background signal from surrounding material. When employing free jet devices, the parabolic flow profile from laminar flow within channels turns into a plug flow profile after passing the nozzle outlet. The liquid–solid interface of the noslip condition resulting in parabolic flow is replaced by the liquid–gas interface in air, which has lower friction with the fluid and can be accelerated in flow direction. Free jets, however, are also quite difficult to work with, and despite the advantages in background have not found widespread use in the field.

#### **2.2 Fabrication**

#### *2.2.1 Fabrication techniques*

Many different technologies exist for the production of microfluidic devices. In general, fabrication of microfluidic devices in hard materials is often very timeconsuming and cost-intensive, thus polymers are generally preferred, particularly when cost and ease of fabrication are considerations in the design process. In most cases, designs are started in silicon, with a Computer-Aided Design (CAD) model of the device. The device is then fabricated using any of a number of different technologies following a process of rapid prototyping. We focus here on some of the more common approaches.


*1 Department of Gene Technology, Vinmec Research Institute of Stem Cell and Gene Technology, Hanoi, Vietnam, 2 Immune Regulation and Cancer, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany, 3 iPS Cell Based Disease Modeling, Berlin Institute of Health, Berlin, Germany*

#### *Edited by:*

*Kun Xu, Northwest A&F University, China*

#### *Reviewed by:*

*Daniela Tropea, Trinity College Dublin, Ireland Wei Li, University of Alabama at Birmingham, United States Robin Ketteler, University College London, United Kingdom*

*\*Correspondence:*

*Van Trung Chu<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics*

*Received: 21 February 2019 Accepted: 17 June 2019 Published: 02 July 2019*

#### *Citation:*

*Le TTH, Tran NT, Dao TML, Nguyen DD, Do HD, Ha LT, Kühn R, Nguyen TL, Rajewsky K and Chu VT (2019) Efficient and Precise CRISPR/ Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells. Front. Genet. 10:625. doi: 10.3389/fgene.2019.00625*

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2R270X mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome.

Keywords: MECP2 mutations, CRISPR/Cas9, RETT syndrome, homologous recombination, iPSCs

#### INTRODUCTION

Rett (RTT) syndrome is a genetic neurodevelopmental disorder found predominantly in female infants. Disease symptoms including cognitive disabilities, decreased coordination and mobility, repetitive movements, and slowed brain growth typically appear after 6 to 18 months of age (Hagberg et al., 1983; Hagberg, 1985; Trevathan and Naidu, 1988). The RTT syndrome is caused by dominant negative mutations in the X-linked transcription factor methyl CpG-binding protein 2 (MECP2). Most mutations occur within one of two functional domains of MECP2: the highly conserved methyl binding domain (MDB) or the transcription repressing domain (TRD) (Rasmussen et al., 1975; Amir et al., 1999). Gene therapy using an adeno-associated viral vector to deliver a functional sequence of the *MECP2* gene *in vivo* has been intensively investigated in mouse models (Sinnett and Gray, 2017). However, this approach induces toxicity and side effects due to the supraphysiological expression of exogenous *MECP2* (Sinnett and Gray, 2017). The study by Guy et al. using conditional MECP2 alleles has shown that by restoring the physiological expression level of *MECP2*, the symptoms of RTT syndrome could be reversed in affected adult mice (Guy et al., 2007; Clarke and Abdala Sheikh, 2018). This study suggests that CRISPR/Cas9-mediated correction of MECP2 mutant alleles is a potential gene therapy for RTT.

The type II CRISPR/Cas9 system is a RNA-guided nuclease providing adaptive immunity in *Streptococcus pyogenes*. In mammalian cells, Cas9 nuclease can be used for editing of genomic sequence by the induction of targeted DNA doublestrand breaks (DSBs). The induced DSBs are mostly repaired by the non-homologous end-joining (NHEJ) pathway creating micro-deletions/or insertions (INDELs) or, to a lesser extent, by the homologous recombination (HR) pathway allowing precise genetic manipulations if a repair template is provided (Cong et al., 2013; Hsu et al., 2013; Mali et al., 2013; Chu et al., 2015). Many studies have shown that CRISPR/Cas9-mediated mutagenesis can lead to efficient HR in human iPSCs (Byrne et al., 2015; Zhang et al., 2017; Li et al., 2018).

To our knowledge, there is no previous attempt to correct endogenous MECP2 mutant alleles in human cells using the CRISPR/Cas9 system. Here, we provide a system to repair the mutation hotspots of the MECP2 exon 4. We achieved HR efficiency up to 20% to 30% in human cell lines and iPSCs. To show a proof of principle of our system, we precisely inserted the MECP2R270X mutation into the MECP2 gene in wild-type iPSCs. Finally, using the CRISPR/Cas9 system, we subsequently repaired this mutation successfully in mutant iPSCs. Overall, we provide an efficient strategy for MECP2 correction that is crucial for disease modeling and can potentially be translated into gene therapy of the RTT syndrome.

### MATERIALS AND METHODS

#### Cell Culture and Reagents

HEK293 cells were maintained in DMEM (Gibco) supplied with 10% FBS (Gibco). K562 cells were cultured in RPMI 1640 supplied with 10% FBS and 2 mM l-glutamine (Gibco). Human iPSCs (BCRT cell line) were cultured in E8 flex medium (Gibco) according to the manufacturer's manual. Cas9 protein was purchased from NEB (M0386S), synthetic sgRNA was purchased from Synthego, and ssODN was ordered from IDT. To generate RNP complex, the Cas9 protein and synthetic sgRNA were mixed at ratio 1:2 and incubated at 25°C for 10 min.

#### CRISPR/Cas9 and Donor Vectors Construction

sgRNAs were designed based on CrisprGold software (Chu et al., 2016). Forward and reverse oligos were mixed and phosphorylated individually. Then, annealed oligo duplexes were cloned into the BbsI sites of the CRISPR/Cas9-T2A reporter plasmid (Addgene, 64216). To generate the pMECP2-T2A-mCherry reporter donor vector, the 5′ and 3′ homology arms (HA) were amplified from genomic DNA using Hercules Phusion polymerase (Agilent). The 5′ HA fragment was cloned into XhoI/EcoRI sites of pTV-T2A-mCherry; the 3′ HA fragment was cloned into AsiSI/KpnI sites of the pTV-T2A-mCherry vector. To generate the pMECP2 donor vector, 5-kb MECP2 fragment was amplified from genomic DNA, and at the cleavage site, the silent mutations were added to generate a new PstI recognition site.

### Transfection, Electroporation, and Flow Cytometry

Human HEK293 cells were plated into six-well plates at 1 day before transfection. On the day of transfection, cells were supplied with fresh complete medium, and the DNA was mixed with FuGENE® HD Reagent (Promega) in Opti-MEM (Invitrogen) according to the manufacturer's introduction. After 15 min of incubation at RT, the mixture was dropped slowly into the well. Next day, the medium was exchanged. The transfected cells were analyzed in different time points. For flow cytometry analysis, HEK293 cells were trypsinized and resuspended in PBS/1% bovine serum albumin (BSA) fluorescence-activated cell sorting (FACS) buffer and analyzed with a Fortessa machine (Becton Dickinson).

For electroporation, human K562 cells were harvested and counted, 2 × 105 cells were resuspended with sgRNA/Cas9 RNP and 100 pmol ssODN in 20 µl electroporation buffer P3 (Lonza) and transferred to a 16-strip cuvette and electroporated using a 4D Nucleofector X unit (Lonza). Then, cells were transferred into the pre-warmed complete medium.

For human BCRT-iPSCs, we used Lipofectamine® 3000 (Life Technologies) according to the manufacturer's manual. Briefly, 1 day before transfection, iPSCs were placed as small clumps in 500 μg/ml of vitronectin (Life Technologies) precoated plate filled with E8 flex completed medium supplemented with Rock inhibitor (10 µM) (Bio Cat). The next day, the medium was replaced without Rock inhibitor 6 to 8 hours before transfection. Diluted plasmids and lipofectamine were prepared as described in manual. In the case of multiple plasmids, pX330 and targeting vector were used for transfection; we used a 1:1 molar ratio. The medium was changed on the next day to stop the reaction. Two days post-transfection, transfected iPSCs were collected by incubation with Accutase (Merck) for 5 min and the cell pellet was then resuspended in a medium containing E8 Flex complete medium, Rock inhibitor (10 µM), RevitaCell Supplement (Life Technologies), and Gentamycin (Lonza) (FACS-PREP medium). GFP+ iPSCs were sorted by FACSAria (Becton Dickinson) and placed into a Vitronectin-precoated plate filled with FACS-PREP medium. The next day, the medium was replaced by E8 flex completed medium. Cells were maintained in culture for several passages before analysis.

For introducing the MECP2R270X mutation into the MECP2 gene, wild-type iPSCs were transfected with 2 μg of the plasmid expressing Cas9 and sgRNA-5 and 60 pmol of ssODN-R270X donor using Lipofectamine® 3000 (Life Technologies). Two days post-transfection, 103 GFP+ iPS cells were sorted and plated on a Vitronectin-coated well of a six-well plate in the FACS-PREP medium. The medium was changed the next day. Seven days after sorting, iPSC clones were picked and plated on a new Vitronectincoated well of a six-well plate. iPSC clones were expanded for 2 weeks. To this end, iPSC clones were harvested for analyzing insertion efficiency by genotyping PCR, RFLP assay, and Sanger sequencing. To repair the MECP2R270X mutation, homozygous mutant iPSC clone (clone 18) was transfected with the plasmid expressing Cas9 and sgRNA-3 together with the donor template plasmid containing silent mutations and a recognition site of PstI restriction enzyme as described above.

#### Genomic DNA Isolation, PCR, T7EI, and RFLP Assay

Reporter+ cells were cultured and harvested at different time points. Single-cell clones were sorted in 96-well plates. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer's instruction. For T7EI assay, PCR was done using Herculase II Fusion DNA Polymerase (Agilent Technology) with PCR gene-specific primers (**Table 1**) using the following conditions: 98°C for 3 min; 39 cycles (95°C for 20 s, 60°C for 20 s, 72°C for 20 s), and 72°C for 3 min. PCR products were run on 2% agarose gels, purified, denatured, annealed, and treated with T7EI (NEB). Cleaved DNA fragments were separated on 2% agarose gels, and the DNA concentration of each band was quantified using the ImageJ software. For genotyping RFLP assay, PCR product was purified and digested with a PstI restriction enzyme for 1 h. The digestion was separated on 2% agarose gels.

DNA sequencing PCR products were directly sequenced by specific primers or cloned into the pSTBlue-1 Blunt vector (Novagen) following the manufacturer's protocol. Plasmid DNAs were isolated using the NucleoSpin Plasmid (Macherey-Nagel). Plasmids were sequenced using T7 forward primer (5′-TAATACGACTCACTATAGGG-3′) by the Sanger method (LGCgenomics, Berlin, Germany).

#### qRT-PCR

Total RNA was extracted from wild-type, mutant, and repaired iPSC clones with RNAeasy Mini Kit (Qiagen) and was reversetranscribed into cDNA with a SuperScript™ III kit (Invitrogen). The expression of MECP2 was measured by real-time PCR using SYBR green PCR Master Mix (Thermo Scientific) and StepOnePlus™ (Applied Biosystems). The relative expression level of MECP2 was normalized with GAPDH housekeeping gene.

#### Statistical Analysis

Statistical tests were performed using Prism 7.0 (GraphPad) using a paired two-tailed Student's t-test. \*\*\*\*P <0.0001.

#### RESULTS

#### CRISPR/Cas9-Mediated Reporter Insertion in the MECP2 Locus

Our previous study identified the mutation spectrum of the MECP2 gene in Vietnamese patients with RTT syndrome. The recurrent mutations T158M, G269fs, R270X, and R306H are located in the MDB and TRD domains of MECP2 protein encoded by exon 4 of the *MECP2* gene (**Figure 1A**). These mutations are specific for the Vietnamese patients, listed in RettBASE (Le Thi Thanh et al., 2018). We first developed a system to quantitatively determine HR efficiency in the human *MECP2* locus by inserting in-frame the coding sequence of cleavage peptide (T2A) and an mCherry reporter in the last exon of the *MECP2* gene. As a result, correctly targeted cells will express the mCherry reporter (**Figure 1B**). We used the CRISPRGold tool (Chu et al., 2016) to design two gRNAs (sg1 and sg2) targeting sequences proximal to the MECP2's stop codon. T7EI assay indicated that both sgRNAs efficiently targeted the *MECP2* locus upon delivery into HEK293 cells along with Cas9. Sanger sequencing showed a broad range of INDELs at the targeted site (**Figure 1C**). To access HR efficiency, plasmids expressing sg1 and Cas9 were transfected into the HEK293 cells together with donor plasmid carrying the MECP2\_T2A\_mCherry repair template. Transfected cells were analyzed by flow cytometry at days 14 and 21 posttransfection. We detected about 23% of mCherry+ cells in HEK293 cells that received sg1, Cas9, and donor template, but only background signals in control groups transfected with sgRNA/Cas9 or donor template alone (**Figure 1D** and **E**). We confirmed the corrected integration of the reporter into the targeted *MECP2* locus by using an external forward primer annealing to a genomic sequence outside of the 5′ HA and a specific T2A sequence reverse primer for PCR. The expected ~3.6-kb fragment was amplified only in HEK293 cells transfected with sg1, Cas9, and donor template (**Figure 1F**). These data indicate the correct configuration of the HR alleles in mcherry+ HEK293 cells. Single mCherry+ cells were sorted into individual wells of 96-well plates for genotyping PCR. We found that about 75% of these cells harbor heterozygous

(above) in the targeted MECP2 sequence. Sequencing data (below) indicates the INDEL spectrum of sg1 in the targeted MECP2 locus. (D) The percentage of mCherry+ cells were analyzed by flow cytometry at days 14 and 21 post-transfection. (E) The HR efficiency was summarized in the graph from three independent experiments, data show means ± SD (\*\*\*\* p<0.0001). (F) Corrected integration PCR. (G) Mono-allelic and bi-allelic knockin analysis in single mCherry+ cell clones. The data represent at least two independent experiments.

integrations of the T2A-mCherry sequence into MECP2, whereas homozygous integrations represent about 25% (**Figure 1G**). Taken together, using CRISPR/Cas9 and reporter systems, we achieved efficient HR in the human *MECP2* locus.

#### CRISPR/Cas9-Mediated Precise Modification in MECP2 Locus

Next, we developed the CRISPR/Cas9-mediated system to correct the recurrent mutations in the MECP2 exon 4: G269fs, R270X, and R306H. We designed two sgRNAs close to these mutations (sg3 and sg4) and a repair template containing HAs of 2.5 kb. To facilitate quantification of the HR efficiency, a PstI restriction site was created by introducing silent mutations into the repair template (**Figure 2A**). High editing activities of sg3 and sg4 were validated in HEK293 cells by T7E1 assays (**Figure 2B**, top panel). Sequencing data of sg3-targeted cells showed a broad range of INDELs (**Figure 2B**, bottom panel). Next, plasmids, carrying Cas9/sg3 and donor template were transfected into HEK293T cells. Thirty days post-transfection, genomic DNA of transfected

quantification-based knock-in efficiency. The data represent at least two independent experiments.

cells was isolated, and the targeted region was amplified by PCR. PstI-mediated restriction fragment length polymorphism (RFLP) showed that PstI-cleaved bands were only detected in cells co-transfected with sg3, Cas9, and repair template. Band quantification showed HR efficiency of 20% to 30% in the *MECP2* locus (**Figure 2C**). Sequencing data of the targeted homozygous clone confirmed that the precise modifications were correctly inserted into the MECP2 gene (**Figure 2D**).

It is known that HR efficiency is determined by many factors. The type of donor template is considered as the most important one (Song and Stieger, 2017). Double-stranded DNA plasmid, PCR sequences, and ssDNA oligonucleotides (ssODN) are often used as a donor template for precise insertion of large or small sequence changes at CRISPR/Cas9-induced DSBs. To test whether we can also use ssODN as donor template for MECP2 precise modifications, we designed a 100-nucleotide ssODN with homology regions of 45 nucleotides each and silent replacements to create a PstI restriction site (**Figure 2E**). Next, we electroporated the sg3/Cas9 RNP complexes with ssODN into human leukemic K562 cells. Four days post-targeting, the targeted cells were harvested for analyzing HR efficiency. As shown in **Figure 2E**, we detected PstI-cleaved PCR products only in cells that received both RNP and ssODN. The HR efficiency ranged from 29% to 34% (**Figure 2E**).

#### CRISPR/Cas9-Mediated Reporter Insertion in Human iPSCs

Patient-derived iPSCs have been intensively studied for disease modeling, drug discovery, and potential somatic cell therapy. Thus, we next tested whether our system works efficiently in human iPSCs. We exploited the reporter system as described above to evaluate HR efficiency. The sg1/Cas9 vector and donor template vector were co-transfected into human iPSCs. Two days later, the transfected iPSCs were enriched and expanded (**Figure 3A**). The transfection efficiency in human iPSCs was about 25% to 28% (**Figure 3B** and **C**). Ten days after expansion, the percentage of mCherry+ iPSCs was analyzed by flow cytometry. As shown in **Figure 3D** and **E**, the percentage of mCherry+ iPSCs was about 20% when transfected with sg1/Cas9 and donor template vectors, whereas there were only background signals in control cells transfected with only sg1/Cas9 or donor

FIGURE 3 | CRISPR/Cas9-mediated reporter insertion in human iPSCs. (A) Experimental scheme of gene editing in human iPSCs. (B) Transfection efficiency in iPSCs using Lipofectamine 3000 was analyzed by FACS. (C) A bar graph indicated the transfection efficiency of human iPSCs, data show means ± SD (\*\*\*\*p < 0.0001). (D) FACS analysis showed the knock-in efficiency in human iPSCs represented by the mCherry+ cells. (E) A bar graph showed knock-in efficiencies in human iPSCs, data show means ± SD (\*\*\*\*p < 0.0001). (F) Corrected integration PCR and Sanger sequencing data showed the junctions of 5′ HA. The data represent at least two independent experiments.

template vectors alone (**Figure 3D** and **E**). Correct integration PCR proved that the T2A-mCherry reporter was successfully inserted into the *MECP2* locus in human iPSCs (**Figure 3F**). Sequencing data confirmed that the targeted MECP2 sequences were configured as planned (**Figure 3G**). Overall, we provide a new efficient system to precisely modify the human *MECP2* gene in human iPSCs.

#### CRISPR/Cas9-Based Modeling MECP2R270X Mutation in Human iPSCs

To precisely introduce a MECP2R270X mutation (c.808 C>T) into the *MECP2* gene in human iPSCs, we designed a new sgRNA (sg5) and ssODN (ssODN-R270X) with homology regions of 60 nucleotides, including the C>T mutation and an XbaI recognition site (**Figure 4A**). Next, we transfected human iPSCs with the sg5/Cas9 vectors and ssODN-R270X donor template. Two days post-transfection, the transfected iPCSs were sorted and single cell-derived clones were expanded (**Figure 4B**). To this end, cell clones were harvested for analyzing the insertion efficiency of the MECP2R270X mutation. Among 22 cell clones, we detected two (9%) homozygous and five (23%) heterozygous clones (**Figure 4C** and **4D**). Sequencing data confirmed that we succeeded to insert the R270X mutation into the *MECP2* gene in human iPSCs (**Figure 4E**). To show a proof of principle of our correction system, we used the strategy depicted in **Figure 2A**. The sg3/Cas9 and donor template vectors were transfected into MECP2R270X/R270X iPS cells (clone 18, see **Figure 4C**). Two days later, single cell-derived clones were isolated and expanded for 2 weeks. As shown in **Figure 4F**, the PstI-mediated RFLP assay showed that we were able to correct the MECP2R270X mutation in mutant iPSCs. To assess the expression of the MECP2 gene, we isolated mRNA from the wild-type, mutant, and repaired iPSC clones. Quantitative RT-PCR data showed that we enabled to restore the expression level of the MECP2 gene in the repaired iPSCs as comparable as the wild-type iPSCs (**Figure 4G**). Sequencing data of MECP2 cDNA confirmed that the mutant MECP2 allele was successfully repaired in the corrected iPSCs (**Figure 4H**). Thus, this system will open new avenues to iPSCbased disease modeling and therapeutic development of RTT syndrome.

### DISCUSSION

In this study, we developed an efficient CRISPR/Cas9-mediated system that precisely modifies the human *MECP2* locus. Restoration of the physiological expression level of the MECP2 gene is critical for the reversion of disease symptoms. Thus, the correction of mutations in the endogenous MECP2 gene holds promise for gene therapy of RTT syndrome. Using preclinical mouse models of RTT, many laboratories have attempted to deliver intact MECP2 cDNA *in vivo,* using the AAV vectors (Sinnett and Gray, 2017). Although AAV-mediated MECP2 delivery leads to positive effects, such as increasing survival and improving weight, this system still causes multiple side effects and toxicity to animals due to the uncontrolled expression level of the MECP2 transgene (Collins et al., 2004; Luikenhuis et al., 2004; Gadalla et al., 2013). Thus, the expression level of MECP2 is essential for restoring the function of defected neurons. Too much MECP2 protein is as harmful to neural cells as too little. It has been shown that patients with the MECP2 duplication syndrome have two copies of the *MECP2* gene on their single X-chromosome leading to mental disability and autistic-like behavior (Moretti and Zoghbi, 2006). Furthermore, a reduction of MECP2 protein due to protein instability is also related to the milder neurological and psychiatric symptoms, such as anxiety and depression (Ramocki et al., 2009; Chao and Zoghbi, 2012). CRISPR/Cas9-mediated mutation correction restores the endogenous expression of the MECP2 gene that might fully recover RTT symptoms. This system can be potentially applied as gene therapy for RTT patients *in vivo*. However, many challenges remain. For example, the mainly affected cells are neurons that are non-dividing. It is known that the HR pathway occurs in the S-G2 phase of the cell cycle. Thus, non-dividing or low proliferating cells, such as neurons, are not suitable for HR-mediated mutant correction. However, this issue can be potentially addressed by either suppressing the NHEJ pathway or activating HR-related factors (Chu et al., 2015; Charpentier et al., 2018). In addition, the safety of the system needs to be further investigated.

In addition, many studies have shown that MECP2 is not only essential for the functions of neurons but also important for the functions of glial subtypes in the brain as well as of many cell types in the immune system, including microglia and macrophages (Maezawa and Jin, 2010; Derecki et al., 2012; O'Driscoll et al., 2013; Cronk et al., 2015; Jin et al., 2015; Cronk et al., 2016). Physiological restoration of normal MEPC2 functions in neurons and in all immune cells should be considered as an optimal therapy. It is supported that hematopoietic stem cell transplantation (HSCT) is beneficial in Mecp2-deficient mice and that MeCP2 plays important roles for immune cells both in the brain and in the periphery (Maezawa and Jin, 2010; Derecki et al., 2012; O'Driscoll et al., 2013; Cronk et al., 2015; Jin et al., 2015). Thus, correction of the MECP2 mutations *ex vivo* with the CRISPR/Cas9 system followed by autologous HSCT is a promising future therapeutic option for RTT syndrome.

Our system also works efficiently in human iPSCs opening great avenues for disease modeling, drug screening, and somatic cell therapy. The generation of RTT patient-derived iPSCs (RTT-hiPSCs) has been reported by many groups; however, the natural random X-chromosome inactivation (XCI) status of RTT-hiPSCs is inconsistent. XCI results in cellular mosaicism where some cells express wild-type MECP2 (isogenic), whereas other cells express mutant MECP2. Some studies showed that the maintenance of inactive X-chromosome of the founder cell allows RTT-hiPSCs to express either wild-type or mutant MECP2 allele (Ananiev et al., 2011; Cheung et al., 2011; Pomp et al., 2011). In contrast, others reported that the inactive X-chromosome of the founder cells is reactivated during the reprogramming process (Kim et al., 2009; Marchetto et al., 2010). Importantly, RTT-hiPSCs could differentiate into neurons that exhibit disease phenotypes of RTT syndrome. Thus, these cells will be valuable for being rescued by CRISPR/Cas9-mediated MECP2

correction. Using CRISPR/Cas9 technology, we have achieved efficient precise MECP2 modifications in human iPSCs. Overall, our work provides an efficient system for repairing MECP2 mutations in RTT-hiPSCs.

#### DATA AVAILABILITY STATEMENT

All datasets generated/analyzed in this study are included in the manuscript or supplementary files.

#### AUTHOR CONTRIBUTIONS

TTHL and VTC designed the project. TTHL, NTT, TMLD, DDN, HDD, TLH, and VTC performed experiments and acquired the

#### REFERENCES

data. NTT, VTC, RK, KR, and TLN analyzed and interpreted the data. VTC, NTT, RK, and KR wrote the article.

#### FUNDING

This work was supported by Vinmec Healthcare System (ISC.17.05 to TTHL and TLN).

#### ACKNOWLEDGMENTS

The authors thank H.P. Rahn for excellent FACS-related support. The authors thank Dr. Harald Stachelscheid from Berlin-Brandenburg Center for Regenerative Therapies for kindly providing human BCRT-iPSC line. The authors would also like to thank K. Petsch and J. Pempe for technical support.

human induced pluripotent stem cells. *Cell* 143, 527–539. doi: 10.1016/j. cell.2010.10.016

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Le, Tran, Dao, Nguyen, Do, Ha, Kühn, Nguyen, Rajewsky and Chu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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Mnar Alnaghes1 \*, Nickolas Falkner1 and Hong Shen2

1 University of Adelaide, Adelaide, SA, Australia

2 Faculty of Sciences, School of Computer Science, University of Adelaide, Engineering, and Technology, Australia

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*An Effective Method for Secure Data Delivery in IoT DOI: http://dx.doi.org/10.5772/intechopen.104663*


*6.3. ISIP-OSOP DC-DC Converter SSM Verification*

In this section, the generalized multimodule DC-DC converter SSM is verified using three different models presented in [38–42].

## 6.3.1. Generalized Model Verification with a Two-Module IPOS DC-DC Converter

The proposed configuration in [38] consists of two modules that are connected in parallel at the input side, and series, at the output side, where the modules are FB-PS-based DC-DC converters. In this model, the effect of the ESR *Rc* is ignored, accordingly, when verifying the model presented in [34] with the generalized model, *Rc* should be equated to zero.

By ensuring ICS and OVS, the input current per module is *Iin* 2 . However, the input voltage per module is *Vin*. Regarding the output side, the output voltage per module is *Vo* 2 . However, the output current per module is *Io*. In which, *Vin* is the IPOS input voltage, *Iin* is the IPOS input current, *Vo* is the IPOS output voltage, and *Io* is the IPOS output current. Accordingly, the values for the six parameters that are previously defined are known and shown in Table 2.

**Table 2.** Values for the six parameters for a two-module Input-Parallel Output-Series (IPOS) power converter.

Substituting the six parameters with their values in Equations (109), (112), (117), and (120) and substituting the ESR *Rc* with zero in the generalized transfer functions would result in the transfer functions presented in Table 3.

**Table 3.** Generalized model verification with the two-module IPOS power converter.

As can be seen from Table 3, the derived generalized model matches the model presented in [38], where the *Rd* term in the above transfer functions is substituted with its equivalence 4 *Llk fs K*2 .

6.3.2. Generalized Model Verification with a Three-Module ISOP DC-DC Converter

In this Section, the generalized model is validated with the three-module ISOP power converter model-derived and presented in [39,40]. The three-module ISOP configuration consists of three modules that are connected in series at the input side, and parallel at the output side. In this model, the effect of the ESR *Rc* is considered, accordingly, when verifying the model presented [39] with the generalized model, *Rc* should not be ignored.

By ensuring IVS and OCS, the input voltage per module is *Vin* 3 . However, the input current per module is *Iin*. Regarding the output side, the output current per module is *Io* 3 . However, the output voltage per module is *Vo*. Accordingly, the values for the six parameters that are previously defined are known and shown Table 4.

**Table 4.** Values for the six parameters for a three-module Input-Series Output-Parallel (ISOP) power converter.

Substituting the six parameters with their values in Equations (109), (112), (117), and (120) would result in the transfer functions presented in Table 5.

**Table 5.** Generalized model verification with the three-module ISOP power converter.

As can be seen from Table 5, the derived generalized model matches the model presented in [39,40].

6.3.3. Generalized Model Verification with a Four-Module ISIPOS DC-DC Converter

In this section, the generalized model is validated with the four-module ISIPOS power converter model presented in [41,42].

The four-module ISIPOS configuration consists of four modules that are connected in series and parallel at the input side, and series at the output side. In this model, the effect of the ESR *Rc* is ignored, accordingly, when verifying the model presented in [41] with the generalized model, *Rc* should be equated to zero.

By ensuring IVS, ICS, and OVS, the input voltage per module is *Vin* 2 and the input current per module is *Iin* 2 . Regarding the output side, the output voltage per module is *Vo* 4 . However, the output current per module is *Io*. Accordingly, the values for the six parameters that are previously defined are known and shown in Table 6.

**Table 6.** Values for the six parameters for a four-module Input-Series Input-Parallel Output-Series (ISIPOS) power converter.

Substituting the six parameters with their values in Equations (109), (112), (117), and (120) would result in the transfer functions presented in Table 7.

**Table 7.** Generalized model verification with the four-module ISIPOS power converter.

As can be seen from Table 7, the derived generalized model matches the model presented in [41,42].


**7.3 Investigating dynamic properties of the spine model under external haptic forces**

Since the finite element spine model for the haptic simulation is represented with beam element which is in fact highly simplified, it is difficult to describe precisely the dynamic properties of the real spine. Compared to this finite element spine model, the detailed musculo-skeletal multi-body spine model developed in LifeMOD can provide better information of biodynamic behaviour of the spine. Since LifeMOD software takes account of other components (such as head, ribcage, muscles, ligaments so on), the locomotion of the spine model will become much more realistic. Furthermore, by simulating the spine model in a haptically integrated graphic environment, the users (such as surgeons, trainers) can quickly and conveniently observe the locomotion as well as gain insight into the dynamic behaviour of the spine.

Development of a Detailed Human Spine Model with Haptic Interface 191

In general, we have presented the process of modelling human spine. Initially, a beam FE spine model for haptic interaction is built based on a solid FE spine model, which is created in offline finite element analysis software. Although there are some limitations on not fully taking into account soft tissues, this beam FE model is able to simulate global dynamic behaviour of the whole spine, Subsequently, an entirely detailed musculo-skeletal mutibody spine model using LifeMOD Biomechanics Modeler is completely developed to solve the limitations aforementioned and reliably validated by comparing simulation results with experimental data and in-vivo measurements. Moreover, to enhance more realistic interaction between users (such as trainers, clinicians, surgeons) and the spine model during real-time simulation, haptic technique is integrated into a graphic interface. Based on this, exploration of the spine model becomes much more realistic since the users can manipulate the haptic cursor to directly touch, grasp and even apply external forces in any arbitrary direction onto any vertebra to observe the deformation of the spine. In such a simulation interface, users can quickly and more conveniently study the locomotion and dynamic

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Gao Z. & Gibson I. (2006). Haptic Sculpting of Multi-Resolution B-Spline Surfaces with

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**8. Conclusion**

behaviour of the spine model.

**9. References**

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The long-term goal of this study is to develop a haptically integrated simulation platform for investigating post-operative personal spine models constructed from LifeMOD. The first step in this study is to develop a haptic interface for a detailed normal spine model whose dynamic properties are obtained from LifeMOD. Figure 26 illustrates the analysis of some dynamic properties of the spine when applying force on vertebra T1 in x-axis direction.

The next step in the study is to develop a post-operative detailed spine model. The operation conducted on the spine model can be spinal fusion or spinal arthroplasty. By using this haptically integrated graphic interface, assessing biomechanical behavior between natural spines and spinal arthroplasty or spinal fusion becomes more convenient. This will help orthepaedic surgeons understand the change in force distribution following spine fusion procedures, which can also assist in post-operative physiotherapy. In addition, the surgeons can gain useful insight from this system in the planning of surgery to correct severe scoliosis. Different designs of rods and braces can for example be experimented with using this virtual environment. It is also considered that this system may assist physiotherapists and other practitioners when designing cushioning and supports for wheelchair-bound and other clients suffering from asymmetric spinal disorders.

Fig. 26. Analyzing translational properties of the spine model under lateral force on T1


Array Pattern Based on Integrated Antenna DOI: http://dx.doi.org/10.5772/intechopen.81087

direction of the antenna element can be changed according to θrad, because the antenna element radiates perpendicularly to the antenna structure. The parameters are given by L ¼ 70 mm, W ¼ 66 mm, lc ¼ 22 mm, wc ¼ 1:5 mm, ls ¼ 24:5 mm, ws ¼ 1:5 mm, lf ¼ 6:2 mm, wp ¼ 8 mm and lp ¼ 32 mm. It is possible to control the impedance matching by modifying l7 and w6. The integrated antenna is produced on a CER-10 substrate with a permittivity of 10 and a thickness of 0.64 mm. The

Figure 4.

transmitting coefficient matrix of the transmit integrated antenna and an N � N identity matrix. The covariance matrix of the MIMO channel H can be described as

Rm� T� 0

P k^r; k ^t � �

� �

. Here, B k^r

is the position of the lth array element and ro is the position of the antenna array

We explained the channel models for the MIMO system with the integrated antenna arrays mathematically. In the following section, we will verify the performance of the integrated antenna array based on the channel model we discussed.

There have been many practical antennas which are designed for the integrated antenna. Through some practical antennas, we can see that the integrated antenna may be implemented for the wireless communication. Design of the integrated antenna is dependent on the system requirement such as array structure and channel environment. In this section, we introduce various practical integrated antennas

It is important to integrate multiple antennas with compact size, which have orthogonal radiation patterns of each other. There are practical integrated antennas without considering array structure as shown in Figure 4. It is found that the integrated antenna has two types of antenna elements which have radiation patterns of electric dipole antenna and magnetic loop antenna. Planer inverted-F antennas (PIFA) are used for the electric dipole antenna, and quarter-wavelength slot antennas are used for the magnetic loop antenna [16, 17]. It is also seen that the integrated antennas have three-dimensional structures because the array structure was not considered. Therefore, it is noted that the integrated antenna may consist of

The integrated antenna array may be implemented as shown in Figure 5. It is found that the practical integrated antenna array is composed of Lt array elements. Each array element consists of Bt antenna elements, which are designed by utilizing

To reduce the antenna size, we eliminated a conduct strip in front of the antenna element and designed that l3 is larger than w2 as shown in Figure 5a. The radiation

⊗R� � �†

m� † n o

B~† k ^r; k ^t � �

¼ er ^ kr � �

, (15)

dΩrdΩt, (16)

¼ ejβð Þ rl�ro k^

, and the E k^

� �

, where rl

. Here, the correlation

⊗A k^r � �

� �

⊗R� � �

m� , m� ¼ vec M� � � and Rm� ¼ E m�

Rh ¼ T� 0

matrix of the channel of the SVW modes can be given by

⊗B k^t � �

is an array element response matrix with the elements of el k^

ðð B~ k ^r; k ^t � �

where h ¼ T� 0

where B~ ^

system.

kr; ^ kt � �

3. Practical antenna design

3.1 Practical integrated antenna

more than two antenna elements practically.

3.2 Practical integrated antenna array

a planer Yagi-Uda antenna [18].

74

for several cases.

⊗R� � �

Array Pattern Optimization

Rm� ¼ 4π2

¼ B k^r � �

Configuration of practical integrated antennas. (a) 6-port integrated antenna and (b) 16-port integrated antenna.

#### Figure 5.

Configuration of practical integrated antenna array. (a) Antenna element for 4-port integrated antenna, (b) 4-port integrated antenna and (c) 4-port integrated antenna based 1 � 4 array.

integrated antenna is expandable to the array structure as shown in Figure 4c, because of the compactness of the integrated antenna.

It is necessary to verify that the integrated antenna has reasonable antenna characteristics such as radiation efficiency, bandwidth and mutual coupling. The proposed four-port integrated antenna was implemented as shown in Figure 6. For the integrated antenna, the antenna elements have the same antenna characteristics of each other because of symmetric configuration. The simulated and the measured S-parameters of the integrated antenna are shown in Figure 7a and b, respectively, where Saa means S1, 1, S2,2, S3, 3 and S4,4; Sab means S1, 2, S1,4, S2, 3 and S3,4; and Sac means S1,3 and S2,4. The integrated antennas radiate at 5.7 GHz with a bandwidth of about 300 MHz satisfying ∣Saa∣ ¼ �10 dB in the simulation and measurement. It is also found that the antenna elements have less than �12 dB mutual couplings in the simulation and measurement.

The simulated and measured S-parameters of the integrated antenna-based 1 � 4 array system are shown in Figure 8a and b, respectively. Only the Sparameters for Port 5 are illustrated because the integrated antenna-based array has

> a symmetric configuration. It is found that the mutual coupling between antenna elements of different array elements is lower by about 13 dB in simulation and measurement results. Thus, it can be noted that the integrated antenna is scalable in

S-parameters of integrated antenna-based 1 4 array. (a) Simulated results and (b) measured results.

found that the simulated and measured radiation patterns are agreed well. It is shown that the antenna elements radiate to y-axis in order to support the front area of the antenna structure and have different radiation patterns. The radiation patterns generated by using the transmitting coefficient matrix T are similar to the simulated radiation patterns as well. Thus, it can be noted that the radiation patterns of the practical antenna can be described in a mathematical expression.

The radiation patterns of the integrated antenna are illustrated in Figure 9. It is

the array structure.

Array Pattern Based on Integrated Antenna DOI: http://dx.doi.org/10.5772/intechopen.81087

Figure 8.

Figure 9.

77

Radiation patterns of proposed four-port pattern antenna.

Figure 6. Prototype of four-port integrated antenna.

Figure 7. S-parameters of four-port integrated antenna. (a) Simulated results and (b) measured results.

Array Pattern Based on Integrated Antenna DOI: http://dx.doi.org/10.5772/intechopen.81087

integrated antenna is expandable to the array structure as shown in Figure 4c,

It is necessary to verify that the integrated antenna has reasonable antenna characteristics such as radiation efficiency, bandwidth and mutual coupling. The proposed four-port integrated antenna was implemented as shown in Figure 6. For the integrated antenna, the antenna elements have the same antenna characteristics of each other because of symmetric configuration. The simulated and the measured S-parameters of the integrated antenna are shown in Figure 7a and b, respectively, where Saa means S1, 1, S2,2, S3, 3 and S4,4; Sab means S1, 2, S1,4, S2, 3 and S3,4; and Sac means S1,3 and S2,4. The integrated antennas radiate at 5.7 GHz with a bandwidth of about 300 MHz satisfying ∣Saa∣ ¼ �10 dB in the simulation and measurement. It is also found that the antenna elements have less than �12 dB mutual couplings in the

The simulated and measured S-parameters of the integrated antenna-based

parameters for Port 5 are illustrated because the integrated antenna-based array has

1 � 4 array system are shown in Figure 8a and b, respectively. Only the S-

S-parameters of four-port integrated antenna. (a) Simulated results and (b) measured results.

because of the compactness of the integrated antenna.

simulation and measurement.

Array Pattern Optimization

Figure 6.

Figure 7.

76

Prototype of four-port integrated antenna.

Figure 8. S-parameters of integrated antenna-based 1 4 array. (a) Simulated results and (b) measured results.

a symmetric configuration. It is found that the mutual coupling between antenna elements of different array elements is lower by about 13 dB in simulation and measurement results. Thus, it can be noted that the integrated antenna is scalable in the array structure.

The radiation patterns of the integrated antenna are illustrated in Figure 9. It is found that the simulated and measured radiation patterns are agreed well. It is shown that the antenna elements radiate to y-axis in order to support the front area of the antenna structure and have different radiation patterns. The radiation patterns generated by using the transmitting coefficient matrix T are similar to the simulated radiation patterns as well. Thus, it can be noted that the radiation patterns of the practical antenna can be described in a mathematical expression.

Figure 9. Radiation patterns of proposed four-port pattern antenna.


*Edited by Jose Luis Rivera-Armenta*

*and Beatriz Adriana Salazar-Cruz*

Asphalt modification is an important area in the development of new road and pavement materials. There is an urgent demand for road materials that can minimize fracture at low temperatures and increase resistance to deformation at high temperatures. The function of asphalt is to bind aggregate to protect it from water and other harmful agents. In the beginning asphalt was ideal for this purpose, but recently traffic loads have increased and environmental factors have deteriorated more rapidly than before. Asphalt is a byproduct of crude oil in the refining process, and it is considered a complex heterogeneous mixture of hydrocarbons. Asphalt modification has become an important research area, using several methods and new materials as modifiers.

ISBN 978-1-78923-726-9

Modified Asphalt

Published in London, UK © 2018 IntechOpen © releon8211 / iStock


Visualisation Approaches

Several improvements have been made to embrace practices related to the visualisation approaches decision category. Currently, the CT offers more precise information regarding the customised units through the customised product prototype (practice 42), and the use of the simplified choice menu to simulate product alternative additional costs (practice 41). During the discussions, the CT architects revealed that they were trying to adapt the choice menu to product type B, in which the range of options is broader than in other product types, highlighting the opportunity of tailoring practices for different market segments.

An intended adoption by the CT can be seen in regard to practice 44 "Virtual prototyping, e.g., building information management (BIM) models, to show product alternatives to customers and ease choice". The initial step was developing three-dimensional models to illustrate product alternatives to be used in meetings with customers.

According to customers, some additional visualisation tools supported decision making during the open days such as standard housing unit prototype, finishing materials catalogue, and, in the third open day, the comparison between the standard and customised housing unit prototypes.

#### 4.2.4. Analysis and Reflection

A low level of implementation of MC practices was identified in Company P, similarly to the results carried out by Fettermann et al. [25] on the MC practices of three Brazilian house building companies. The main improvement opportunities identified in this investigation were also similar to that study, being concerned with the solution space and visualisation decision categories, and customerbased knowledge decision domain. Jensen et al. [2] argue that by understanding customer's needs and preferences and making product recommendations based on the available solution space, companies can save much time in the configuration process, and also increase quality and reduce rework. Additionally, the implementation of MC practices enabled Company P to provide a better service for customers and to improve efficiency in some internal processes.

The CT has pointed out in the discussion meetings that some practices could be adapted for other product types that had a higher degree of customisation. However, this would require the analysis of a different context, in which the complexity of interactions with stakeholders would be much higher. These considerations reinforce the need for devising context-specific practices and implementation guidelines, as suggested by Suzic et al. [20].

The lack of communication, according to Andújar-Montoya [9], can be attributed to the fact that the NPD in housing is often divided into stages, which are not properly integrated. Beyond that, Schoenwitz et al. [60] suggest that this disconnection reflects different degrees of awareness regarding the customisation strategy, similar to what was observed in Company P. For example, the sales department was willing to extend the list of options with the aim of signing a contract, in opposition to the production management team. This often occurs due to different mindsets, concerns and nature of the job [60]. According to CT members', this reflects the lack of a common understanding in Company P of the role and impacts of customisation in house building projects. Thus, by encouraging better communication between departments, companies should be able to build up relationships based on trust, mutual commitment and understanding of others expectations, which might avoid extra costs and delays [9].

According to Gherardi [34], a shared understanding is needed to apply MC practices, i.e., a minimum agreement is necessary for the practice to be adopted and continue to be used. Therefore, there must be opportunities for increasing the awareness of di fferent stakeholders regarding MC, as well as for negotiation when deciding to adopt MC practices as a way to promote innovation.

#### *4.3. Assessment of the Utility of the Solution*

The utility of the proposed decision categories and the list of practices was tested in two di fferent stages of the empirical study, both in terms of identifying improvement opportunities and assessing the evolution of the MC strategy. The decision categories were also used to increase the CT awareness and understanding regarding key concepts, enabling them to provide numerous examples and opinions during the discussions. In fact, the CT stated that through the discussions they were able to perceive underlying ideas that they overpass in daily routine, and that this can also be useful as arguments when discussing with other departments, which contributes to improve collaboration.

The discussions regarding practices and decision categories were also useful to understand the scope of the MC strategy of Company P, and, more specifically, to identify gaps and limits for implementation. For instance, some of the solution space practices were immediately rejected by the CT, due to limitations of MC scope that were defined by existing capabilities, and focus on specific market segments. Moreover, the customer and customisation manager highlighted that the practices identified in this investigation could be useful to support decision making, such as, for refining the solution space based on the choice of users from previously delivered residential projects. Furthermore, the participants pointed out the need to improve the identification of customers' needs and to provide feedback to NPD as two major gaps in the MC strategy of the company, highlighting the importance of the customer based knowledge decision domain.

Several improvement opportunities provided further evidences of the utility of the customer integration and core decision categories. After the first presentation of research findings, many improvements were undertaken, regarding the communications with other departments, customer interaction and relationship, and visualisation approaches. Furthermore, the refinement of the strategy was also influenced by lessons learned from other segments, projects and experiences. An example is the simplified choice menu that was adopted for some market segments, in which the team had more experience. That successful solution inspired the customisation department to adapt it to A and B product types. This example reinforces the need for creating a knowledge system that enables continuous improvement and organisational learning.

The discussions with the CT also brought to light many relevant customer integration aspects. The CT coordinator highlighted the utility of customer integration decision categories in terms of making explicit what the company o ffers, and how the customer is involved, which makes the decision-making process as straightforward as possible. In fact, some practices related to product visualisation approaches that were implemented by the company along the study, such as the choice menu, the customised product prototype, and 3D models had a positive impact in terms of explaining the solution space to customers.