**2.3 Comparison between different reconfiguration techniques**

Electronic switching components have been widely used to reconfigure antennas, especially after the appearance of RF-MEMS in 1998. One of the main advantages of such components is their good isolation and low-loss property. While RF-MEMS represents an innovative switching mechanism, its response is slower than PIN diodes and varactors, which have a response on the order of nanoseconds. The ease of integration of such switches into the antenna structure is matched by their nonlinearity effects (capacitive and resistive) and their need for high voltage (RF-MEMS, varactors). The activation of such switches requires biasing lines that may negatively affect the antenna radiation pattern and add more losses. The incorporation of switches increases the complexity of the antenna structure because of the need of additional bypass capacitors and inductors, which increase the power consumption of the whole system.

**Figure 1.** *Antenna reconfiguration techniques [4].*

### *Array Antenna for Reconfigurations DOI: http://dx.doi.org/10.5772/intechopen.106343*

Even though optical switches are not much famous, they offer a reliable reconfiguration mechanism especially in comparison to RF-MEMS. The activation or deactivation of the photoconductive switch by shining light from the laser diode does not produce harmonics and intermodulation distortion due to their linear behavior. Moreover, these switches are integrated into the antenna structure without any complicated biasing lines, which eliminates unwanted interference, losses, and radiation pattern distortion.  **Table 1** shows a comparison of the characteristics for the different switching techniques used on electrically and optically reconfigurable. The advantages of using physical reconfiguration techniques lie in the fact that they do not require bias lines or resort to laser diodes or optical fibers. However, their disadvantages include slow response, cost, size, power source requirements, and the complex integration of the reconfiguring element into the antenna structure. As for the materially reconfigurable antennas, one major advantage of using liquid crystals is their moderate losses and ease of tuning. Graphene is one other tunable wonder material, which exhibits excellent properties. At submillimeter wave where the footprint of antenna is very small and integrating switches is not possible; we can use these tunable materials to make reconfigurable antennas.

Reconfiguration is possible at GHz as well as THz frequencies as per requirement and applications. Nowadays, advance communication in 5G or more than 5G requires high frequency, which can give high bandwidth for wide range uses. In **Tables 2** and **3**, lists of reconfigurable antennas are given, which are already designed and in use.

The state of the art of research on material-based reconfigurable antennas at GHz frequencies from **Table 2** shows that only few LC materials are explored to achieve reconfiguration. At microwave frequency, graphene is very less used due to high loss. Even LC materials show moderate losses at microwave frequency but because of fabrication limitations, LC is preferred over graphene at microwave frequency. Graphene is much more used than LC at THz frequencies due to unique effect of surface plasmon polariton (SPP) wave. Frequency reconfiguration range depends on the range of permittivity achieved with different LC materials. The designed antenna is based on K15 with no reported gain in literature as given 1 to 0 dBi [24, 25, 29]. A novel design of microstrip patch antenna is proposed based on K15 LC [28]. This antenna has 6.2 dB of gain with good impedance matching. The designed antenna is based on E7 LC reported in [26]. The reported gain is 0.7–1.1 dBi, whereas the gain is not reported [27]. The designed antenna based on GT3-23001 LC is proposed in [30] with the designed structure of 1 4 element antenna array with 12 dBi gain. The antenna applies a double-layer structure with no reported gain [31]. The designed

#### **Table 1.**

*Electrical properties of electrically and optically switching [4].*

**Table 2.** *Comparison of reconfigurable antennas at GHz frequency reported in the literature.*

antenna is based on transparent DLA 100-100 LC with poor impedance matching as reported in [32]. Gain of this structure is not reported. The designed antenna is based on graphene with gain 0.76–2.38 dB in [32] and 0.1–1.9 dB in [33, 34]. At microwave frequency, biased graphene works as a lossy metal, and hence, gain of the antenna reduces due to high power dissipation and increased sheet resistance. So, based on the above literature review, LC is chosen over graphene for the proposed S (2–4 GHz) band antenna for WLAN/WiMAX applications. Comparative analysis of antenna performance and tuning range is done with different LC materials.

The state of the art of research in material-based reconfigurable antennas at THz frequencies from **Table 3** shows that at higher frequency, graphene is preferred over LC. Property of spp waves in graphene help in extreme miniaturization of the antenna size with good performance. Most of the antennas designed in 0–1 THz band are given in [4, 35–43, 45]. The graphene-based microstrip patch antenna is designed on silicon substrate with gain 2.43–4.19 dB, efficiency 48–51%, and return loss 13–26 dB [35]. The graphene-based microstrip patch antenna designed on glass (SiO2) substrate with capacitive-coupled transmission line is given in [36]. Reported gain is 5.08 dB, efficiency is 66%, and return loss is 39.19 dB. The graphene-based patch antenna is designed on silicon substrate with efficiency 16–29%, return loss 23–30 dB, and no reported gain [37]. The graphene-based microstrip patch antenna is designed on polyimide substrate with gain 4.93–5.07 dB, return loss up to 35 dB, and efficiency 86.53–6.85% reported in [4]. The graphene-based Yagi-Uda antenna is designed on glass (SiO2) substrate with gain 6.5 dB and return loss 19 dB [38]. The graphene-based patch antenna designed on glass (SiO2) substrate with directivity 2.99–5.56 dB and return loss 24–34 dB is given in [39]. The graphene-based 1 2 microstrip patch antenna array designed on glass (SiO2) substrate with efficiency 8–43%, return loss 16–30 dB, and directivity 9 dB is given in [40]. The graphene-based patch antenna is

#### **Table 3.**

*Comparison reconfigurable antennas at THz frequency reported in the literature.*

designed on GaAs substrate with efficiency 20% and no reported gain [41]. The graphene-based microstrip patch antenna is designed on Duroid substrate with return loss up to 40 dB and no reported gain [45]. The graphene-based square spiral antenna is designed on quartz substrate with return loss up to 37 dB, impedance bandwidth 26.68%, and no reported gain [42]. The LC-based reflect array (with 54 52 multiresonant cells) in which LC is inserted between quartz and silicon is designed on FR-4 with gain 19.4 dB and return loss 13 dB [43]. So, based on the literature review shown in **Table 3**, it is summarized that graphene-based antennas are much more suitable than LC at THz frequencies. Most of the graphene antennas proposed till now are based on either thick layer of graphene or multilayer graphene with moderate performance. Mostly frequency reconfiguration is reported, very few on radiation pattern, and almost none on polarization reconfiguration. The proposed antenna array is based on monolayer graphene with frequency, polarization, and radiation pattern reconfiguration all together with good performance.


**10. LCHF and non-alcoholic fatty liver disease (NAFLD)**

Non-alcoholic fatty liver disease (NAFLD) is characterised by elevated TG and low HDL-C concentrations with overproduction of VLDL and impaired clearance of TG-rich lipoproteins [94, 95]. It is also recognised that cardiovascular disease is the leading cause of death in NAFLD [96]. It has been shown that NAFLD with insulin resistance is the cause of atherosclerotic disease characterised by many of these features [97]. Since NAFLD is caused by excessive carbohydrate, especially fructose intake [98–100], it is postulated that a carbohydrate-restricted LCHF diet can reverse NAFLD.

using fats. However, these symptoms may be especially prevalent only in the period of adaptation to the diet, after which most subside. Some suggest additional sodium (especially for cramping) and fluid intake to minimise side effects, since excretion of water and sodium are

Low-Carbohydrate High-Fat (LCHF) Diet: Evidence of Its Benefits

http://dx.doi.org/10.5772/intechopen.73138

81

Some have suggested that weight loss on LCHF diets is the result mainly of water loss. This increased diuresis may be true in the first weeks of carbohydrate restriction [104]. However, body composition by DEXA analysis indicates that long-term weight loss on the LCHF diet is

Trials show that adherence to LCHF and LFHC diets are similar [13, 72, 105]. On the other hand, a recent systematic review found a higher attrition rate from LFHC than from LCHF diets [106]. Therefore, sticking to a LCHF diet is perhaps as convenient as any other dietary plan and thus may be more sustainable as it tends to reduce hunger without need for specific calorie restriction. In fact, studies of long-term adherence of up to a year [38, 39] on the LCHF

From the current evidence and above reviews of lower carbohydrate diets, it can be postulated that LCHF diets reduce insulin resistance, improves glycaemic regulation and has positive effects on reducing cardiovascular risk factors, including reducing serum triglyceride, increasing HDL cholesterol, increasing LDL particle size and reducing blood pressure. A substantial proportion of individuals have also been shown to discontinue one or more

Low-carbohydrate high-fat (LCHF) diet has been shown to be as effective as other diets for weight reduction, through increased satiety and reduction in calorie intake. LCHF diet also helps improve glycaemic control in type 2 diabetes mellitus and in otherwise healthy patients

Some of the benefits of the LCHF diet results from the often large weight loss typically produced by this diet. Therefore, at least some of the beneficial changes from LCHF diet would also be experienced by patients prepared to adhere to any calorie-restricted diet. LCHF diets control energy balance through increased satiety and reduced ad libitum energy intake while encouraging the ingestion of a nutrient-dense diet by replacing refined foods with natural foods.

LCHF diets have beneficial effects on cardiovascular risk factors through their effect on blood lipid concentrations. They decrease triglycerides, apoprotein B and saturated fat levels in

increased on these diets as a result of reduction in insulin levels with LCHF diet [103].

predominantly the result of the loss of fat mass with some loss of fat-free mass [3].

**11.5. Weight loss on LCHF diets is due to increased water loss**

**11.6. Sustainability**

**12. Summary**

diabetes medication.

with insulin resistance.

have not identified any evidence of harm.

Thus, LCHF diet is likely to benefit patients with high TG to HDL-C ratios and NAFLD, all of which are common in the insulin-resistant individual. A recent lifestyle intervention trial reduced the prevalence of metabolic syndrome from 58 to 19% among obese and overweight patients treated with LCHF for 3–8 months, showing how quickly carbohydrate restriction can improve health in those with metabolic syndrome [37].


**5.3. Finding the photoelectric characteristics of solar cells**

integrand *f*(*x*,*V*) and its half-width Δ*x*1/2. By calculating Δ*x*1/2 as the difference of the values for which *f*(*x*,*V*) is equal to ½, one can obtain under conditions that *qV* approaches not very close

j

Eq. (56)

*qV E qV qV E qV* (55)

*qV E qV kT* (56)

Eq. (51)

 m

 m

bi g bi g

So Eq. (53) in the integral form yields the exponential dependence of the recombination current

m

Fig. 21 shows a comparison of the voltage dependence of the recombination currents in CIGS

exp 1 . ( )( 2 ) 2

0 0.2 0.4 0.6 0.8

*V* (V)

**Figure 21.** Comparison of the recombination currents in CIGS solar cell with 1.14 eV bandgap calculated from Eqs. (51)

As seen in Fig. 21, the *J–V* curves practically coincide. Currents calculated from the exact Eq. (51) exceeds those calculated from Eq. (56) by no more than 5–6% at low voltages and no more than 8–10% at higher voltages. When *V* > 0.7 V the curves are separated from one another

é ù æ ö » - ê ú ç ÷ - -D- ë û è ø

*qn W kT qV J*

solar cell, for example, with *E*g=1.14 eV calculated using Eq. (51) and (56).

2 2arccosh2 2 . 2( ) 2 ( )( 2 )

D » ç ÷ = - -D- - -D- è ø

*E qV kT x W W*

to *E*g – 2Δμ:

and (56).

1/2

m

1/2

on the applied voltage (sinh(*qV*/2*kT*)=exp(*qV*/2*kT*)/2) [37, 38]:

 j

104

102

10–2

*J*

(mA/cm

)

2

100

10–4

no po bi g

æ ö -D-

tt

i

j

g

38 Solar Cells - New Approaches and Reviews

First consider an applicability of the above theory of generation–recombination in the SCR to the experimental data discussed in Section 5.2.

The dark *J–V* curves (circles) along with the calculated results (solid lines) using Eq. (51) are shown in Fig. 22. In the calculations, we used the exact Eq. (51) in order to reflect the deviation of the forward current from the expression *J* ∝ exp(*qV*/2*kT*), when *qV* approaches *φ*bi. For CIS solar cell (*E*g=1.04 eV) the experimentally obtained data is shown without modification (shunting is virtually absent), whereas for CIGS with the absorber bandgaps 1.14 and 1.36 eV the presented experimental data is obtained by subtracting the current through the shunts *R*sh and taking into account the voltage drop across the series resistance *R*s. As mentioned previously, at *V* < 0.1–0.2 V, the values of current under illumination *J*IL and the short-circuit current *J*sc are very close to each other, therefore the dark current cannot be determined with a proper accuracy and the experimental points are not shown for low voltages for the three solar cells. For solar cell with bandgap of the absorber *E*g=1.5 eV fabricated at elevated temperature the measured data of the dark current is shown for low voltages as well.

**Figure 22.** Comparison of the dark forward characteristics of CIGS solar cells extracted from the *J–V* curves under illu‐ mination [8] and the generation current (solid lines) calculated from Eq. (51).

As seen in Fig. 22, the calculated results agree well with the experimental data for all solar cells. One point to be mentioned is that in order to obtain a fit with the experimental data of solar cell with *E*g=1.5 eV fabricated at elevated temperature, the effective carrier lifetime in the SCR was taken about an order of magnitude less than that for other solar cells. This explains why the curves with so much different bandgaps, *E*g=1.36 and 1.5 eV, are located close to each other. It should also be emphasized that reducing the carrier lifetime results in the lowering of open-circuit voltage by ∼ 0.06 V, which apparently is one of the negative aspects of the growth and post-growth processing at elevated temperature [36].

Knowing the dark *J–V* characteristics and the short-circuit current densities, it is not difficult to calculate the *J–V* curves under illumination as the dependences of *J*IL=*J – J*sc vs. *V*, which are shown in Fig. 23a by bashed lines. For the short-circuit current densities we use the data given in Fig. 18a, although they can be obtained using the spectral distribution of the quantum efficiency and solar radiation (for the studied samples the discrepancy between the *J*sc values calculated by these two methods does not exceed 3–4%). Also shown in Fig. 23a by circles are the measured results of the *J–V* curves for the samples with the absorber bandgaps 1.04, 1.14 and 1.36 eV.

Fig. 23b shows the dependences of the electrical power in the solar cell circuit *P*=(*J – J*sc)*V* as a function of voltage *V*. The solid lines in both figures are the calculated results corresponding to the presence of shunts in cells, whereas dashed lines are the results obtained by subtracting the current through the shunt from the measured current.

35

much different bandgaps, *E*g = 1.36 and 1.5 eV, are located close to each other. It should also be emphasized that reducing the carrier lifetime results in the lowering of open-circuit voltage by 0.06 V, which apparently is one of the negative aspects of the growth and post-growth processing at elevated

Knowing the dark *J–V* characteristics and the short-circuit current densities, it is not difficult to calculate the *J–V* curves under illumination as the dependences of *J*IL = *J – J*sc vs. *V*, which are shown in Fig. 23a by bashed lines. For the short-circuit current densities we use the data given in Fig. 18a, although they can be obtained using the spectral distribution of the quantum efficiency and solar radiation (for the studied samples the discrepancy between the *J*sc values calculated by these two method does not exceed 3–4%). Also shown in Fig. 23a by circles are the measured results of the *J–V*

Fig. 23b shows the dependences of the electrical power in the solar cell circuit *P* = (*J – J*sc)*V* as a

curves for the samples with the absorber bandgaps 1.04, 1.14 and 1.36 eV.

temperature [36].

*E*g *=* 1.36 eV

*E*g *=* 1.5 eV

*E*g *=* 1.14 eV

0 0.2 0.4 0.6 0.8

*V* (V)

**Figure 22.** Comparison of the dark forward characteristics of CIGS solar cells extracted from the *J–V* curves under illu‐

As seen in Fig. 22, the calculated results agree well with the experimental data for all solar cells. One point to be mentioned is that in order to obtain a fit with the experimental data of solar cell with *E*g=1.5 eV fabricated at elevated temperature, the effective carrier lifetime in the SCR was taken about an order of magnitude less than that for other solar cells. This explains why the curves with so much different bandgaps, *E*g=1.36 and 1.5 eV, are located close to each other. It should also be emphasized that reducing the carrier lifetime results in the lowering of open-circuit voltage by ∼ 0.06 V, which apparently is one of the negative aspects of the

Knowing the dark *J–V* characteristics and the short-circuit current densities, it is not difficult to calculate the *J–V* curves under illumination as the dependences of *J*IL=*J – J*sc vs. *V*, which are shown in Fig. 23a by bashed lines. For the short-circuit current densities we use the data given in Fig. 18a, although they can be obtained using the spectral distribution of the quantum efficiency and solar radiation (for the studied samples the discrepancy between the *J*sc values calculated by these two methods does not exceed 3–4%). Also shown in Fig. 23a by circles are the measured results of the *J–V* curves for the samples with the absorber bandgaps 1.04, 1.14

Fig. 23b shows the dependences of the electrical power in the solar cell circuit *P*=(*J – J*sc)*V* as a function of voltage *V*. The solid lines in both figures are the calculated results corresponding to the presence of shunts in cells, whereas dashed lines are the results obtained by subtracting

*E*g *=* 1.04 eV

10–2

100

10–4

10–6

mination [8] and the generation current (solid lines) calculated from Eq. (51).

growth and post-growth processing at elevated temperature [36].

the current through the shunt from the measured current.

*J*

and 1.36 eV.

(mA/cm

)

2

40 Solar Cells - New Approaches and Reviews

102

illumination *I*IL (a) [8], and the electrical power *P* in the circuit of solar cell (b). The measured results are shown by circles, the results of calculations considering the presence of shunts and without them are shown by solid and dashed lines, respectively. **Figure 23.** Comparison of voltage dependences of the measured current in CIGS solar cells under illumination *I*IL (a) [8], and the electrical power *P* in the circuit of solar cell (b). The measured results are shown by circles, the results of calculations considering the presence of shunts and without them are shown by solid and dashed lines, respectively.

Figure 23. Comparison of voltage dependences of the measured current in CIGS solar cells under

As seen in Fig. 23a, the calculated curve for sample with the bandgap 1.04 eV practically coincides with experimental curve, but in the cases of the absorbers with bandgaps 1.14 and 1.36 eV the calculations give overestimated values of the current. However, if the shunts are taken into account, the calculated and experimental curves for all samples practically coincide. It follows that a comparison of the theory and experiment gives the *quantitative* information on the electrical losses in solar cells. These losses manifest themselves clearly on the voltage dependences of the electrical power *P* produced by solar cell, *P* = (*J – J*sc)*V* shown in Fig. 23b (as in Fig. 23a, the solid lines are the calculated results with the presence of a shunt in cell, whereas dashed line are the results obtained by subtracting the current through the shunts from the measured currents.). As seen in Fig. 23a, the calculated curve for sample with the bandgap 1.04 eV practically coincides with experimental curve, but in the cases of the absorbers with bandgaps 1.14 and 1.36 eV the calculations give overestimated values of the current. However, if the shunts are taken into account, the calculated and experimental curves for all samples practically coincide. It follows that a comparison of the theory and experiment gives the *quantitative* information on the electrical losses in solar cells. These losses manifest themselves clearly on the voltage dependences of the electrical power *P* produced by solar cell, *P*=(*J – J*sc)*V* shown in Fig. 23b (as in Fig. 23a, the solid lines are the calculated results with the presence of a shunt in cell, whereas dashed line are the results obtained by subtracting the current through the shunts from the measured currents.).

As seen in Fig. 23, the calculated results agree well with the experimental data and indicate a noticeable negative effect of shunting in the studied cells. The effect of shunting is higher when the bandgap of the CIGS absorber is large (shunting does not reveal itself in CIS solar cell). As expected, the shunting does not practically vary the open circuit voltage but reduces the fill factor and the energy conversion efficiency. For the cell with absorber bandgap 1.14 eV, shunting leads to decreasing the fill factor from 0.73 to 0.70 and the efficiency from 13.3 to 12.7%. For cell with the absorber bandgap 1.36 eV, these values are 0.77 to 0.70 and 14.3 to 13.0%, respectively. The fill factor and efficiency of CuInSe2 solar cell are equal to 0.67 and 11.3%, respectively.

Note that the efficiency of studied CIGS solar cells is in the range of 11-14% which is comparable with the efficiency of the modules produced in large volume, but much inferior to record efficiency of small area CIGS solar cells achieved so far.


**9. Orchestrating Anti-Cancer Innate Immune Cells in the Absence of Functional HLA**

A great strength of oncolytic viruses is that they are fully customizable drugs that can be fine-tuned to accomplish the tasks at hand. Where adaptive immunity is unlikely to be harnessed, arming elements targeting cancer vaccine approaches can be swapped out for those targeting the innate immune system. NK cells have been directly implicated in enhancing the efficacy of oncolytic viruses in experimental models [83]. Arming oncolytic viruses with IL-15 [84] is likely to augment NK activation, while strategies based on CCL5 chemotaxis [85] have been used to attract them in to tumors. Expression of SIRPa-Fc antagonists from oncolytic viruses can block the CD47 "don't eat me" signals on cancer cells and facilitate macrophages to attack them directly [86]. Building on the BiTE principle, it is now possible to develop bispecific killer cell engagers (BiKEs) that crosslink cancer cell surface antigens with CD16 on NK cells, or better still, trispecific TriKEs that included further functionality through integrating IL-15 [87,88]. Equivalent approaches using BiMEs (bispecific macrophage engagers) to activate macrophages have also been reported [89]. Like all arming approaches to activate the adaptive immune system, strategies for exploiting innate cells will benefit from selective expression in the tumor microenvironment provided

by oncolytic viruses. Careful design of the innate immune cell stimulation is required in order to avoid the premature and unwanted elimination of virus-infected cells.


*Edited by Luis Rodrigo*

The term "ascites" is from the Greek word askites meaning "baglike." Although most commonly due to cirrhosis, severe liver disease or metastatic cancer, its presence can be a sign of other significant medical problems, such as Budd-Chiari syndrome. Diagnosis of the cause is usually done with blood tests, an ultrasound scan of the abdomen, and direct removal of the fluid by a needle or paracentesis (which may also be therapeutic). Treatment using medications (diuretics), external drainage, or other treatments is clearly defined. In this book, the authors describe the physiopathology of the diverse causes of ascites,the types of treatments recommended, the recent advances achieved, the complications and the prognosis of the different clinical situations that doctors must face.

ISBN 978-953-51-3613-2

Ascites - Physiopathology, Treatment, Complications and Prognosis

Photo by Ugreen / iStock

## Ascites Physiopathology, Treatment, Complications and Prognosis

*Edited by Luis Rodrigo*


3.2.2. Hand-Type Nozzle

Similarly to the cannon-type nozzle, the three studied droplet size variables in the hand-type nozzle also depend on the HP, LFR and AS (*p* < 0.001). This fact is consistent with the air speed decrease found in the hand-type nozzle (Figure 4). General droplet size interval for every tested position can be observed in Figure 5b. Droplet size was more heterogeneous in the XP compared to that observed in the cannon spout.

The D50 values measured for the hand-type nozzle (Figure 7a) were slightly higher than those obtained in the cannon spout. Thus, CP resulted in D50 below 150 μm, with the minimum mean value of 62 μm for the combination between minimum LFR and maximum AS. XP, on the other hand, resulted in D50 values above 125 μm, with a maximum value of 407 μm for the combination of minimum LFR and minimum AS. In this particular case, the atypical behavior already observed for the cannon was also registered in the hand-type spout, with a decrease in the droplet size with the increase in the LFR. There was also a considerable influence of the other two evaluated parameters (*p* < 10−3) on D50. In the case of LFR, there was a positive effect on D50, whilst the opposite was found for AS, as expected from previous works [53]. The mean D50 increase that can be found between CP and AP was 42%. From AP to OP there was a mean increase of 39%. Last, OP to XP had a mean VMD increment of 75%.

**Figure 7.** (**a**) D50 (Volume Median Diameter, VMD), (**b**) D10 and (**c**) D90 values (μm) per liquid hose position (HP), liquid flow rate (LFR) and airflow speed (AS) for the hand-type nozzle. Bars show the mean ± Standard Error. CP, conventional insertion of liquid hose position; AP, alternative position; OP, out position; XP, extreme position.

The D10 values (Figure 7b) comprised a minimum value of 23 μm for the combination of CP, intermediate LFR and maximum AS, and 179 μm for the combination of XP, minimum LFR and

minimum AS. The CP presented D10 values all below 50 μm, while the XP presented values above 55 μm. The mean increase in this parameter from CP to AP was 49%, from AP to OP it was 3% and from OP to XP it was 135%.

The D90 values (Figure 7c) were all found below 280 μm in CP and above 370 μm in XP. The most extreme values were 112 μm (for CP/min LFR/max AS) and 735 μm (for XP/min LFR/min AS). The mean increase from CP to AP was 35%, from AP to OP it was 28% and from OP to XP it was 94%.

In general, it can be observed that the most extreme positions generated an atypical response of the LFR on the droplet size parameters. Nevertheless, the three LFR levels did not produce great di fferences in comparison with the AS ones. It could be stated that LFR had a lesser impact on droplet size in the outermost positions of the liquid hose.

#### *3.3. Cumulative Sprayed Volume Curves and Their Classification According to ASABE S572.1*

The cumulative sprayed volume curves, obtained with cannon (Figure 8) and hand-type (Figure 9) nozzles by testing all configurations (combination of HP, LFR and AS), compared with the American Society of Agricultural and Biological Engineers (ASABE) nozzles classifications (ASABE S572.1) [63], showed that an appropriate selection of pneumatic sprayer operational parameters, namely LFR and AS, led to a limited change in the spray quality generated, varying from very fine (VF) to fine (F) in the best cases. On the contrary, changing HP deeply a ffected the spray quality and reached the coarse (C) (Figure 8) and very coarse (VC) (Figure 9) spray quality when the liquid hose was tested in the extreme position (XP) combined with reduced AS, irrespective of LFR. Even if Balsari et al. [53] recognize the importance of a proper selection of LFR and AS parameters in order to vary the spray quality in conventional pneumatic sprayers, likewise it was clear that the spray quality extent change from VF to F was not enough to guarantee environmental safeguard, despite the increased droplet size dimension. For this reason, according to the preliminary tests performed by Miranda-Fuentes et al. [55], the HP change along the pneumatic spouts is proven as the driving factor for changing the spray quality. This strategy allows to achieve a range of di fferent spray qualities comparable to that reachable with hydraulic nozzles, which can be varied in type (conventional vs. air induction) and size [43]. Thus, at the time of application, in both pneumatic nozzle types the selection of HP allowed to vary the spray quality in a wide range without changing the other operational parameters selected for the application (LFR and AS). In detail, the cumulative sprayed volume curves derived from the tested configurations using the liquid hose in conventional position (CP) showed a droplet spectrum similar to the hydraulic nozzles Albuz ® ATR lilac used at 0.7 MPa (Figures 8 and 9) and selected as reference. Only the hand-type nozzle tested in CP position with AS of 59.7 and 64.6 m s−1 showed a coarser spray quality, namely fine (F), irrespective of LFR (Figure 9). On the contrary, none of the tested configurations with both cannon- and hand-type pneumatic nozzles in XP position achieved the extra coarse (XC) spray quality, likewise that generated by the air-induction Albuz ® TVI8001 nozzles (0.7 MPa pressure) used as reference nozzle for the coarse spray quality (Figures 8 and 9). In general, the liquid hose position in AP and OP allowed intermediate spray quality, varying from F to M according to the LFR and AS selected. Even when the hand-type nozzle generated averagely larger droplets than the cannon-type did, the spray quality produced by both of them, for the same tested configuration, was very similar (Figures 8 and 9). In practice, no substantial di fference in prospective coverage of the leaves and fruits could be noticed between the two nozzle types. In any case, the possibility to vary in a wide range the droplet size by simply adjusting the liquid hose position gives, for the first time, the opportunity to both farmers and technicians to match the environmental requirements, balancing at the same time the treatment specifications for every spray application while using pneumatic spraying in vineyards. Recently, Grella et al. [58] demonstrated through field trials that the variation in the spray quality over the range investigated (from CP to XP) did not a ffect the canopy coverage, while coarser sprays (liquid hose in AP and XP) produced greater deposits on the target. Concurrently, the use of cannon spout in XP position significantly reduced the o ff-field ground losses in the downwind area.

**Figure 8.** Cumulate sprayed volume (%) curves as a function of droplet size (μm) per liquid flow rate (LFR), airflow speed (AS) and liquid hose position (HP) for the cannon-type nozzle. In each graph the hydraulic reference nozzles hollow cone conventional ATR lilac and air-induction TVI 8001 (Albuz®) are displayed, both operated at 0.7 MPa pressure. CP, conventional insertion of liquid hose position; AP, alternative position; OP, out position; XP, extreme position. VF, very fine; F, fine; M, medium; C, coarse; VC, very coarse; XC, extremely coarse; UC, ultra-coarse/unclassified; (ASABE S572.1).

**Figure 9.** Cumulate sprayed volume (%) curves as a function of droplet size (μm) per liquid flow rate (LFR), airflow speed (AS) and liquid hose position (HP) for the hand-type nozzle. In each graph the hydraulic reference nozzles hollow cone conventional ATR lilac and air-induction TVI 8001 (Albuz®) are displayed, both operated at 0.7 MPa pressure. CP, conventional insertion of liquid hose position; AP, alternative position; OP, out position; XP, extreme position. VF, very fine; F, fine; M, medium; C, coarse; VC, very coarse; XC, extremely coarse; UC, ultra-coarse/unclassified; (ASABE S572.1).

## *3.4. Droplet Homogeneity*

Significant di fferences (*p* < 0.001) were found in both nozzles for every single variable and their interactions. In general, it could be stated that the distance increase to the CP generates an increase in the droplet heterogeneity (Figure 10). This result is in line with the results obtained in previous works for the CP and the AP [55]. RSF values were slightly lower in the hand spout than in the cannon one (1.63 vs 1.78), especially in the surroundings of the CP.

In the cannon spout, the hose position change, although statistically significant, did not generate major di fferences in the mean RSF values (Figure 10a). Thus, values for every single position were near 2.00. The observed trend was a reduction in the droplet population homogeneity with the distance increase from the CP. The increase in AS also increased the RSF in general, especially in the case of the lower LFR in the CP (Figure 10a).

There was a much higher influence of the hose insertion position in the case of the hand spout (Figure 10b). In this case all values were around 2.00, but with important di fferences among the tested hose positions. There was a significant particularity in the hand spout case: values presented a higher RSF in the OP than in the XP, while the opposite would have been the most predictable scenario (Figure 10b). It should be pointed out that in this nozzle both LFR and AS had a higher impact on the RSF than they had in the cannon spout.

**Figure 10.** Relative SPAN Factor (RSF) per liquid hose position (HP), liquid flow rate (LFR) and airflow speed (AS) for both the (**a**) cannon-type and (**b**) hand-type nozzles. Bars show the mean ±Standard Error. CP, conventional insertion of liquid hose position; AP, alternative position; OP, out position; XP, extreme position.

The comparison between both nozzles regarding the droplet homogeneity follows a trend similar to that observed in previous works [53]. In those, a positive influence of both LFR and AS was observed on the RSF in both nozzles. Similarly to the present findings, the cannon spout had a higher RSF than the hand-type one did. A marked increase in the droplet heterogeneity was also observed in the maximum AS and minimum LFR combination (Figure 10a). This could be explained by the fact that with a very low LFR the amount of sprayed liquid might be insu fficient for the high air current to produce an optimal water division into homogeneous droplets, thus increasing the amount of very fine ones, altering their normal distribution and increasing the droplets heterogeneity by a ffecting the kurtosis.


**4.2 Rapid sequence induction and intubation**

To minimize the risk of aspiration, rapid sequence induction and intubation (RSI) has become the standard induction technique in obstetric general anesthesia [2, 60–62]. The goal of RSI is to minimize the length of time between loss of consciousness and the establishment of optimal intubating condition. In the traditional RSI technique, after achieving ideal patient position, preoxygenation, and application of cricoid pressure, the administration of an intravenous induction agent is then rapidly followed by administration of an intravenous neuromuscular blocking agent; the trachea is intubated without attempts at positive-pressure ventilation (PPV) [3, 60]. The goal of avoiding PPV is to prevent gastric insufflation, as this may increase the risk of regurgitation [63]. However, OAA/DAS recommends considering the use of facemask ventilation, as the risk of regurgitation is low with correct application of cricoid pressure. Therefore, the steps in performing RSI in obstetric patient are: (1) proper patient positioning; (2) preoxygenation; (3) application of cricoid pressure; (4) administration of induction agent and neuromuscular blocking agent; (5) face mask ventilation; (6) tracheal intubation [49].

In obstetric procedures, propofol is the typical induction agent, barring hemodynamic or respiratory concerns. After the administration of induction agent, neuromuscular blocking drugs (NMDs) are then administered intravenously to facilitate optimal intubating condition. Succinylcholine or suxamethonium has historically been the most commonly used NMD for RSI in obstetric patients because of its rapid onset and short duration, allowing resumption of spontaneous ventilation in the event of failed intubation [2, 3, 64]. The optimal dose of succinylcholine is 1–1.5 mg/ kg [3, 65–67]. Although the level of plasma pseudocholinesterase decreases during pregnancy, studies show that the duration of action of succinylcholine in pregnant women remains unchanged [68]. Since the advent of sugammadex, a fast-acting reversal agent specifically for rocuronium or vecuronium, nondepolarizing NMDs (e.g., rocuronium, vecuronium) can also be used in RSI technique as the alternative to succinylcholine [64, 69, 70]. The optimal dose of rocuronium is 1–1.2 mg/kg, and vecuronium is 0.3 mg/kg [71–75]. Sugammadex (16 mg/kg) can rapidly reverse the effects of rocuronium or vecuronium to prevent the prolong duration of action. Therefore, it can achieve the same clinical effects as succinylcholine without risk of hyperkalemia, bradycardia, myalgia, increased intragastric pressure, and increased intracranial pressure [71–76].

In contrast to standard RSI, modified RSI with low-pressure ventilation (<12 cm H20) with correct application of cricoid pressure, is recommended in RSI for obstetric patients. Ventilation is carried out using a facemask with a maximum inflation pressure of 20 cm H2O. The goal of PPV is to delay the onset of hypoxemia and increase the likelihood of successful facemask ventilation in the event of difficult or failed tracheal intubation [77, 78].

In pregnant women without risk of difficult airway, direct laryngoscopy using a Macintosh blade is preferred [2, 3, 79]. However, in patients at risk of difficult airway (e.g., obese) and those who fail tracheal intubation, practitioners should use a video laryngoscope to provide a better view of the glottis and thereby increase the chances of success [80–90]. Since all obstetric patients are at a higher risk for a difficult airway, video laryngoscope should always be available in all obstetric general anesthetics [49, 79]. If the view of the glottis is not optimal during the first laryngoscopy, the anesthesiologist may reduce or remove cricoid pressure and reposition the head and neck [91, 92]. Due to capillary engorgement and edema of the mucosal lining of the

airway in pregnant women, the risk for airway obstruction and bleeding during upper airway manipulation is higher than in the general population [13, 14]. Therefore, a small endotracheal tube (e.g., size 6.5 or 7.0) is recommended to minimize the risk of trauma and increase the success rate in pregnant women. To minimize the risk of pulmonary aspiration, a cuffed endotracheal tube is used. In addition, endotracheal tube introducer (e.g., flexible stylet, bougie) can be used to improve the success rate of tracheal intubation [2, 49].


**8. Time tested tips for breeding for drought across crops**

A deluge of information and knowledge has been generated over time. Considerable invest‐ ment has been made by the international community and funding agencies. The collective intelligence can be summarized as under.

if stage specific drought resistance is built into a variety and should drought occur at some other stage. Breeding effort would be futile.

**9.** Need to select for combination of stresses that challenge the crop in the farmers' field rather than only drought resistance. This will ensure longer survival of the variety in the farmers' field [22].

**Figure 4.** Borrowed from Kell DB, Annals of Botany, pp 1-12.[23]

In conclusion, breeding for drought resistance can do only as much as develop a genotype that can tolerate to moisture stress and respond to incremental water inputs should that be possible in the given habitat. The final answer to maximizing productivity comes from an integrated approach where genotype, agronomy, management, economics and policy come together to maximize the water productivity, the key limiting natural resource. Water is not equitably distributed in the world and the scarcity of water is assuming ominous dimensions (Figure 3).

A deluge of information and knowledge has been generated over time. Considerable invest‐ ment has been made by the international community and funding agencies. The collective

**1.** Screen parents and segregants in a habit that most closely represent/ resemble the target

**2.** Repeat phenotyping over years and seasons so that maximum possible variations in the

**3.** Impose selection starting from early generation (not F2) in well-watered and stress conditions. This will ensure selection for potential productivity (should stress not occur in the farmers' fields) and still maximize productivity under stress (should it occur). This will ensure that the farmer is likely to harness the full potential of every incremental drop

**4.** Quantify the water that is received by the field during the experimental period. Input of water could come in the form of rainfall or surface irrigation. It would be ideal to also document the evapotranspiration from the field. This will help budget water at the level of each genotype/segregant/plot. With such data, water productivity could be computed

**5.** While stability of the drought variety is highly desirable, local adaptability is also very important. A genotype may manifest low stability and may be use-full for the particular

**6.** High yield under favorable conditions (moisture regimes) can be combined with the high degree of drought resistance. Thus, traits associated with drought need not be a penalty

**7.** Selection for root and shoot morphology can be judiciously combined in ongoing breeding

**8.** Instead of incorporating growth stage specific drought resistance (vegetative stage drought resistance, reproductive stage drought resistance etc.) it is more appropriate to make any and every growth stage of the crop drought resistant as the timing of occurrence of drought, if at all in the farmers' field is unpredictable. It would be counterproductive

**8. Time tested tips for breeding for drought across crops**

intelligence can be summarized as under.

environmental conditions are represented.

of water above the threshold called drought.

to the plant under well-watered condition.

program while selecting for resistance to diseases and pests.

for each segregant, genotype.

habitat.

population of habitats.

162 Plant Breeding from Laboratories to Fields

This calls for ushering a blue revolution [2, 23], the water equivalent of green revolution, where it is envisaged that the water productivity in agriculture is addressed along with equity of water distribution between urban aspirations and rural needs. Finally [24] proposes that roots do much more than supply water to plants, they capture carbon and a well-endowed root system is not only important to the plant but also as a means to mitigate climate change, by trapping carbon in the form of increased microbial activity and dry matter accumulations. Thus, roots not only provide anchor, water, nutrients but also contribute to carbon sequestra‐ tion and one of the means to mitigate global warming.

Finally, using all shoot and root characters of the plant, be it morphological, physiological, biochemical, phenological, anatomical or responses to environment would provide more opportunities for enhancing drought resistance of the crop. The characters of the plant have to be matched with the appropriate agronomic practices to maximize the expression of the traits. This is a holistic approach to plant breeding for drought resistance and also referred to as 'whole-plant breeding'.


*Edited by Feng Shi*

The main target of this book is to state the latest advancement in ceramic coatings technology in various industrial fields. The book includes topics related to the applications of ceramic coating covers in enginnering, including fabrication route (electrophoretic deposition and physical deposition) and applications in turbine parts, internal combustion engine, pigment, foundry, etc.

ISBN 978-953-51-0083-6

Ceramic Coatings - Applications in Engineering

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**3. MRI principles**

MRI is based on the concept of nuclear magnetic resonance. Atomic nuclei with an odd number of protons or neutrons exhibit a net spin entailing a charge circulation that forms an individual magnetic field surrounding the atom, giving the protons a

magnetic dipole [50–55]. As the hydrogen atoms exhibit ±½ spin, and the nuclear spins exist in two states that are randomly oriented, in absence of a net magnetic field, there is no overall net magnetization. When placed in an external magnetic field, the spins orient parallel and anti-parallel to the direction of the B0 magnetic field, with a slight propensity for the spins to align in the parallel direction, causing the tissue to express a net equilibrium magnetization [50, 53]. The magnetic moment of the atom rotates like a spinning top, predominately in the direction of the applied magnetic field. This magnetic moment rotates at an angular frequency unique to individual atoms, termed the Larmor frequency.

When a perpendicular radiofrequency field (B1) is applied at the hydrogen Larmor resonance frequency, only the protons absorb energy, and are tipped from the direction of the main magnetic field, with the flip angle denoting the degree that the spins are displaced from the equilibrium B0 direction [55]. This excites the protons to precess in a rotational motion around the B0 field vector. The excited proton magnetization vector then relaxes in the direction of the main B0 magnetic field, generating a longitudinal and transverse time-varying magnetization signal that is detected by the MRI receiver coils.

The rate at which this magnetization vector relaxes towards the main magnetic field direction is measured in terms of spin-lattice relaxation (T1) in the direction of the B0 magnetic field, and the spin-spin relaxation rate (T2) trans-verse to the B0 magnetic field direction [50, 53]. The T1 and T2 decay rates result from random static magnetic field variations. However, the relaxation rates are also influenced by time varying factors, such as magnetic field inhomogeneities, that combine with tissue static magnetic field to affect the relaxation rate.

The net magnetism applied to each proton results from both the field generated from the MRI system, in addition to the fields generated by the surrounding protons and bulk susceptibility [55, 56]. A chemical shift in the precession frequency results from the magnetic fields generated from these surrounding protons. This can allow identification of specific molecules present in the tissue, that introduce a distinctive chemical shift in the MR signal [55]. The degree of this shift also has a temperature dependence. Using the principle of proton resonance frequency shift (PRFS) thermometry, the individual temperature of each voxel can be quantified from the resulting temperature-dependent phase change due to this chemical shift [56].

### **4. MRI liver imaging**

Radiological imaging is used in a variety of manners in treating CRLM: including, to diagnose a condition, stage the disease, to locate extra-hepatic metastases, for treatment planning, for interventional image-guided procedures, and for post-treatment evaluation [57]. MRgHIFU requires additional MRI sequence protocols, compared to general diagnostic MRI.

### **4.1 Diagnostic MRI for CRLM**

Although CRLM is usually confirmed with computed tomography, MRI is an acceptable and common alternative, and is advantageous at identifying small lesions [1]. Some studies have shown MRI to provide the best results among all diagnostic imaging modalities, though more expensive [58]. The primary objectives for MRI liver tumor diagnosis are to verify the neoplasm presence, staging the lesion, and

### *Magnetic Resonance-Guided Focused Ultrasound in the Treatment of Colorectal Cancer Liver… DOI: http://dx.doi.org/10.5772/intechopen.105906*

classifying the type of neoplasm [22]. Accurate assessment of these techniques is crucial to guiding subsequent treatment such as resection, biopsy, and chemotherapy [22]. National Comprehensive Cancer Network (NCCN) guidelines recommend CT be used for initial workup and staging; with MRI recommended for potentially resectable cases, prior to locoregional treatment, and for inadequate imaging with CT [59].

Metastatic liver tumors have been reported as a factor of 18–40 more frequent than primary tumors [17]. The presence of both benign and malignant liver lesions are common. The challenge is often distinguishing the benign liver lesions from malignant lesions, as misdiagnosis can greatly impact staging and treatment planning. CRLM lesions exhibit T1 signal hypointensity, higher FATSAT-T2W signal intensity, and higher diffusion-weighted imaging (DWI) signal intensity. On T2W, the tumor resembles a target; with coagulative necrosis causing a relatively higher signal intensity in the tumor center, followed by a reduced signal exterior due to bulk desmoplasia, and an even lower intensity thin edge from desmoplasia growing at the periphery. This thin edge resembles a ring in the arterial phase when gadolinium is administered. These features can change due to fatty liver infiltration and edema [60].

Standard liver tumor protocols are concerned with imaging the parenchyma, vascular supply, and biliary tract [61]. Basic liver protocols often include: T2 half acquisition single-shot turbo spin echo (HASTE) localizer, in-op phase T1 Gradient Recall Echo (GRE), T2 fast spin echo (FSE) with fat saturation (FATSAT), and gadoliniumenhanced 3D FATSAT T1 GRE [61, 62]. The HASTE localizer uses a motion insensitive T2 single-shot spin echo sequence in combination with half-Fourier to acquire a multislice image in about 2 seconds during a single breath hold [63].  The spectroscopic method distinguishes an image at the ∙CH2 fat chemical shift from an image at the water chemical shift [64]. In-phase and op-phase sequences are often spin-echo or GRE sequences with equal repetition times, but different echo times. It acquires a normal in-phase image containing the water and fat, an opposed-phase image containing the water phase signal lessened by the fat phase contribution. Combining in-phase and op-phase images generates the water only image, and subtraction of the op-phase image from the in-phase image allows isolation of the fat signal [64, 65]. Additionally, the Dixon technique allows the generation of a T2 \* map, from which the local iron content (mg g−1) can be formulated [65, 66].

Of high importance in clinical diagnosis of liver lesions are DWI and hepatocytespecific magnetic resonance contrast agent imaging, with MRI elastography to a lesser extent [67]. DWI is particularly useful for detection of small metastatic lesions [61]. Liver DWI consists of a T2 sequence with symmetric diffusion sensitizing gradients centered on the 180° refocusing pulse [67, 68]. Brownian motion of water molecules is more restricted in tumors and provides a noticeable degree of contrast compared to normal tissue [69]. The DWI sequence is generally used without the administration of a contrast agent, making it a completely non-invasive diagnostic sequence. The weighting factor in DWI is adjusted based on the b-value, that is a function of gradient strength and duration. The apparent diffusion coefficient (ADC) maps can be viewed by removing the T2-weighting from a series of diffusion-weighted images. Hyperintense regions generally correspond to regions of low fluid diffusion [63].

CRLM lesions are a solid liver lesion and a general protocol for identification and characterization can be described as follows. First, a highly T2-weighted SSTSE to identify benign fluid-filled lesions, such as cysts and hemangiomas. Next, a modestly T2-weighted FATSAT-TSE or DWI to identify metastatic tumor sites. Then, a Dixon

sequence might be used to observe the degree of fat infiltration into the tumor. Lastly, a contrast-enhanced image can be used for T1-weighted phase imaging to characterize the tumor [70].

Extracellular gadolinium agents are the most common contrast agents for general imaging throughout the body [71]. Two common hepatic specific contrast agents are gadoxetate disodium (Gd-EOB-DTPA, Primovist, Eovist, Bayer Healthcare Pharmaceuticals) and gadobenate dimeglumine (Gd-BOPTA, Bracco Diagnostics) [67]. In some studies, Gd-EOB-DTPA hepatocyte specific MRI contrast agents has shown improved sensitivity and specificity in diagnosis of liver metastasis compared to computed tomography, particularly to the improved ability to detect small metastases [67, 72]. The hepatic-specific contrast agents are specific to tumors originating from hepatocytes, and can help distinguish these lesions from cavernoma or metastatic lesions [71, 73]. Though, these are more expensive than extracellular analogues, have a lower recommended dose and signal, and can exhibit reduced uptake in patients with hepatocyte dysfunction [69]. A comparison of DWI and Gd-EOB-DTPA-T1W MRI for detecting small lesions from CRLM are shown in **Figure 1** [74].

### **4.2 MRgHIFU sequence aspects**

MRgHIFU requires additional MRI sequences that allow for temperature mapping. MR temperature mapping most commonly utilizes PRFS thermometry [75–77], though other possible techniques allow temperature measurements based on the temperaturedependence of relaxation rates, proton density, water diffusion coefficient, thermosensitive contrast agents, and magnetization transfer [78, 79]. Resonance frequency shift results from temperature differences in water molecules and aqueous tissues, due to varying degrees of hydrogen bonding. At increased temperatures, the amount of hydrogen bonding is reduced. This increases nuclear shielding of water protons from the incident magnetic field, generating a lower resonance frequency in the water molecules [78]. This results in a linear-dependence of the phase map values from the chemical shift due to temperature change, at a rate of about −0.01 ppm °C−1 [78, 79]. MRgHIFU sequences

### **Figure 1.**

*Comparison of diffusion-weighted MRI with contrast-enhanced T1W MRI. Left: diffusion-weighted MRI of liver metastases. The arrows indicate small metastatic tumors, less than 1 cm diameter. Right: CE-T1W image after applying Gd-EOB-DTPA, in the same patient. Reprinted with permission from Koh and Berry [74].*

### *Magnetic Resonance-Guided Focused Ultrasound in the Treatment of Colorectal Cancer Liver… DOI: http://dx.doi.org/10.5772/intechopen.105906*

are often based on GRE segmented echo-planar imaging (SEG EPI) sequences. A basic GRE sequence is the fast low-angle shot (FLASH) sequence that utilizes small flip angles to obtain a short echo time (TE) and repetition time (TR) [80]. The sequence further benefits from EPI to accelerate the acquisition rate.

Additional sequences are used to assess tissue peri-ablation and post-ablation. During the peri-ablation period, inflammation in the focal region results from edema, giving more contrast enhancement, and remains for some months. After ablation, T2 and peripheral T1 hyperintensity increases significantly due to the presence of hemorrhagic debris at the ablation region. Thickening or nodule formation in the peripheral hyperintense signal can also indicate recurrence or incomplete ablation, during the months following the procedure [67]. Alternative sequences are under study for other aspects of the modality. For example, magnetic resonance acoustic radiation force impulse (MR-ARFI) sequences allow simultaneous displacement and temperature measurements [81, 82], and is implemented in clinical research settings for tracking focal spot and assessing positioning errors [83, 84]. Additionally, MR-ARFI sequences are being studied for phase aberration correction that occurs in transcostal or transcranial procedures [85–87].

Also, thermal ablation needs temperature processing less than about one second. The faster sequences result in reduced signal to noise ratio and increased temperature uncertainty. Echo planar imaging, parallel imaging, alternate trajectories, and undersampling can increase the MRI frame rate [88–90]. In typical rectilinear sampling, the RF pulse frequency and slice-select gradient determine the slice to be imaged, the frequency encoding gradient amplitude controls the kx-dimension position, and the phase encoding gradient amplitude controls the ky-dimension position [54]. Alternate trajectories are useful, particularly for fast acquisition times and reducing motion artifacts. Radial trajectories are utilized in some of the fastest real-time MRI sequences [90]. Magnetic field inhomogeneities and magnetic susceptibility are also significant aspects to proper imaging and temperature mapping [91, 92].


**4. Discussion**

This study demonstrated that the penetrating spring-loaded CBE device can be a useful technique in the humane euthanasia of experimental animals by successfully producing instant and irreversible unconsciousness in 100% of the guinea pigs. This assessment of loss of consciousness is based on the absence of reflexes and the significant palpable physical damage to the skull, indicating sufficient damage to the vital regions of the brain [5,16,53]. The small but consistent result in this study exceeds the accepted animal welfare standard proposed by Temple Grandin for livestock in abattoirs, whereby 95% of animals should be immediately stunned with a single shot [54]. It also further supports the results from the study conducted by Limon et al. on South American commercial guinea pigs and is comparable to findings for other vertebrate species. In this study, some guinea pigs displayed involuntary muscle fasciculations and slow hindlimb pedaling motions after the initial shot. Both pedaling and head/neck movements are known autonomic movements controlled by reflex circuits in the spinal cord; thus, they commonly occur even after spinal severance due to the residual activity of these circuits [7,53,55].

The CB device used in this study was purchased from a commercial supplier which advertised it as a penetrating CB device. From the above results, all the skulls had extensive fractures, which is characteristic of a penetrating CB, although the skin remained intact after the procedure. This appearance is more characteristic of a non-penetrating CB which has a concussive effect without the bolt entering the cranium or penetrating the skin. Hence, while the skin remained intact, this paper refers to the device as a penetrating CB based on the photographs post-euthanasia as the bolt has clearly entered the cranium. An unexpected outcome of the experiment was that the overlying cranial skin of all guinea pigs was not compromised and remained intact. This observation was different to what was anticipated, especially given the extent of the fractures observed, and is not a typical characteristic of a penetrating CB. The intact skin may potentially be the result of the increased elasticity of the skin around the head in a guinea pig compared to the skin of other animals, such as cattle and rabbits, where the skin is more tightly adhered to their skulls. The cranial fractures associated with the position of the CB on the skull was expected given the nature of the device. The combination of the absence of measured parameters and post-mortem examination (hemorrhages and fractures) demonstrated that CBE resulted in effective stunning and insensibility in all the guinea pigs studied, even though the overlying skin was not compromised.

### *4.1. E*ff*ectiveness of Captive Bolt in Animals*

Currently, there is only one published review of CBE in guinea pigs. This study was performed on South American commercial guinea pigs and four different slaughter methods were compared (cervical dislocation, electrical head-only stunning, carbon dioxide stunning and penetrating CB) [13]. Similarly to this paper, a small sample size of guinea pigs was used (10) with one guinea pig requiring a second shot. This could be argued as a 10% fail rate which is above the Temple Grandin recommendations for humane euthanasia with CB. Possible reasons for this guinea pig requiring a second shot may have been related to handling and restraint, incorrect placement of the CB, differences in devices or device failure. These problems have been described in other studies investigating CB in other species, in which insufficient pressure and/or penetration of the device, inaccurate placement of the device, inappropriate CB chosen for the intended species or prior head injury in animals were associated with failure of CBE or the need for multiple attempts [10,20,23,26,28]. These are all aspects to be managed and explored appropriately when using CBE. It is noteworthy that the use of CBE in our study for experimental tissue collection did not result in failure for any animal involved, despite the relative inexperience of the researchers with this technique.

The findings from these papers, along with this present study, support the conclusion that CBE can be an effective and humane method of euthanasia across many vertebrate species. In contrast, BFT has been shown to have a comparatively high failure rate. BFT also has the potential to more frequently fail the international standard of having at least 95% of animals being stunned after an initial blow/strike to preserve animal welfare. Therefore, when compared to CBE, BFT has a higher potential to negatively impact the welfare of guinea pigs and other vertebrate animals. The use of CBE appears to support the 3R framework principle of refinement by utilizing a more humane and preferable method to BFT in animals used for research.

### *4.2. Uncontaminated Tissues*

At times, high quality chemically uncontaminated non-brain tissues are required for medical or biomedical research purposes. According to the operators in this study, the tracheal tissues collected were of a higher quality compared with tissues obtained from previous experimental work utilizing the BFT method. In these types of experiments, the majority of other euthanasia options available for guinea pigs (see Table 1) are not ideal as they have the potential to contaminate tissues, decrease tissue quality and affect metabolic serum biomarkers [45,46]. These issues can be potential confounders that can affect or compromise experimental outcomes, which could mean the information obtained may be incorrect or inconclusive. As such, the use of CBE over BFT supports the 3R principle of reduction as fewer animals may be required when higher quality tissues are used with better results. By utilizing high quality tissues, fewer animals may be used as research outcomes which may be more robust with a lower rate of rejected tissues. In regards to guinea pigs raised for human consumption, there is a legal and ethical requirement to ensure that meat products do not have any chemical residues unsuitable for human consumption and that animals are humanely killed. The use of CBE can be seen as a preferred method to support refinement due to potential improvements in animal welfare and research outcomes whenever intact brain or nasal tissues are not required [5].

### *4.3. Physical, Emotional and Compassion Fatigue*

Operators anecdotally reported increased satisfaction with using a CBE compared to BFT due to the decreased variability of the effectiveness and easier application of the technique. This could potentially reduce the likelihood of physical, emotional and compassion fatigue being experienced by the operators. This is similar to statements made by other operators in different experiments which also rated CBE as the most aesthetically pleasing method of euthanasia when compared to BFT [8,17,22]. Aspects of operator fatigue include physical, emotional and compassion fatigue. Physical fatigue can be reduced when the CB is correctly placed on the cranium as the kinetic energy is delivered by the device [56,57]. This is different to the use of BFT which relies on the force applied by operators.

Emotional and compassion fatigue can also be linked to the human–animal bond. This bond is thought to be a contributing factor in the compassion fatigue experienced by people working in the animal profession [29]. Thus, by reducing the strain on the human–animal bond, both emotional and compassion fatigue can possibly be reduced. In this study, the operators using the CB reported this method to be more aesthetically appealing, as the bolt did not appear to significantly compromise the skin (Figures 3 and 4). They also felt the method of euthanasia was more humane. As a result, this method appeared to be less confronting and overall more appealing to operators, and potentially less detrimental to their wellbeing.

While the human element is an important aspect of humane euthanasia, the psychological aspect of this euthanasia technique was not objectively assessed in this paper. However, the initial anecdotal evidence appears to be positive. In future studies, a survey could be used to assess the emotional effect of CBE use to further investigate the human welfare aspect of various techniques of humane euthanasia. Overall, there was a verbalized positive impact on the physical and psychological well-being of operators when using this method in this case study. However, for CBE to be successful in future experimental studies or settings, adequate training and correct restraint of the animals must be ensured, and an appropriately sized CB device must be used and maintained. Other causes for CBE failure can arise if personnel are not adequately trained, which can lead to incorrect restraint of animals and placement of the device. If any of the above are performed incorrectly, higher levels of physical and emotional fatigue can occur due to failure of the device or technique [5,7–9,20]. Therefore, correct training and device usage is essential to decrease the chances for physical, emotional and compassion fatigue, as well as reducing risks to personnel and animals [3,36,58].

### *4.4. Limitations and the Future*

Due to the small sample size of guinea pigs, aspects such as device fatigue and physical fatigue experienced by operators from loading the spring with each guinea pig were not evaluated. Other potential parameters that determine insensibility and success of stunning include measuring auditory evoked potential (AEP) and electroencephalogram (EEG) activity with electrodes. Both correlate to the cessation of convulsions, loss of reflexes and brain death [8,59,60]. These were not used in this study as the five parameters for humane livestock slaughter were deemed adequate to indicate insensibility. The use of EEG in CBE also risks damaging the electrodes and was deemed inappropriate for this study [8].

Additional parameters to measure could include the absence or presence of a heartbeat. However, cardiac contractions can continue in unconscious or brain-dead animals and thus this parameter was not used [8,18,61]. Whilst histological changes or scoring of the damage to the cranium and brain could have been assessed (skull fractures were easily observed and palpated), the degree of brain and cranial damage was not considered essential in this study [8,13,17,18]. An area that would benefit from additional investigation would be to explore alternate types of restraint and equipment. The current technique described requires a handler and a CB operator. It would be beneficial to develop or modify a device to enable the task to be performed by only one CB operator.

Finally, a di fferent type of CB device could be tested. The use of an industrial CB (gunpowder-powered, compressed air or CO2 blank cartridges) may be more suitable than the spring-powered CB in larger cohorts or in an abattoir setting for animals bred for human consumption. This is because the spring-loaded CB is designed to be used for <1000 animals per year and can experience device fatigue more quickly compared to an industrial CB [51]. The downside of an industrial CB is that they are more expensive and require more comprehensive training to use. However, the use of an industrial CB can result in less physical fatigue compared to a spring-loaded CB as there is no requirement to physically pull the spring with each use. Thus, the use of industrial CBs could be considered if a greater number of animals are to be used in a study due to larger cohorts or in other settings (i.e., an abattoir). It should also be noted that as of 2020, within Australia, CB is an accessible humane euthanasia technique as there are no legal requirements to hold a firearms license when using a CB in six out of the seven Australian states and territories. The authors are aware of similar legislation in other countries, which makes CBE a potentially accessible and humane euthanasia technique internationally in research and other settings.

From this study and other studies that have been undertaken, it can be tentatively proposed that CBE can be used as a suitable alternative for the collection of non-brain uncontaminated tissues. A drawback of this study is the small sample size available at the time. Further research should be undertaken to review the potential failure rate on a larger population to determine potential complications. Future studies in refinement could explore di fferent handling/restraint techniques or devices, and the e ffects of the use of di fferent CBs. Additional work can be performed to ensure consistency of the results from this study. As compassion and emotional fatigue are so prevalent, any future studies should incorporate formal surveys of operator preferences regarding their levels of physical, emotional and compassion fatigue and how this changes when performing euthanasia in di fferent conditions.


**6. Future directions**

Another important molecular interactor of STIM1 is the hypoxia-inducible factor-1 alpha (HIF-1α), which is upregulated during hepatocarcinoma growth [85]. Li et al. found that HIF-1α directly controls STIM1 transcription, but also that STIM1-mediated SOCE is required for HIF-1α accumulation in hepatocarcinoma cells via activation of Ca2+/CaM-dependent protein kinase II, revealing a mutual dependence of STIM1 and HIF-1α in the regulation of Ca2+

High levels of ORAI1 and STIM1 are found in many types of cancer cells. In gastric cancer tumor progression, this higher expression is associated with a negative impact on survival rates of patients, an effect that was partially due to targeting expression of metastasis-associated in colon cancer-1 (MACC1) [86], an essential regulator of the transcription for the gene coding for the hepatocyte growth factor receptor, MET. Similarly, a recent report described that STIM1 promotes cell migration and the epithelial-to-mesenchymal transition (EMT) by acti-

Finally, an excellent report from Stephan Feske laboratory [54], described how SOCE is crucial for mitochondrial fatty acid oxidation, and that Ca2+ entry through ORAI1 was essential to activate adenylyl cyclase, cyclic AMP production, the transcriptional regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and peroxisome proliferator-activated receptor alpha (PPARα), which is mediated by the activation

Shortly after the molecular description of ORAI1 as the CRAC channel, it was revealed the essential role of other ORAI proteins in cell signaling. The involvement of ORAI3, together with ORAI1, in arachidonic acid-regulated Ca2+ (ARC) channels was early proposed [88, 89]. In contrast to SOC channels, the activation of ARC channels depends on the pool of STIM1 resident in the plasma membrane [90]. More interestingly, ORAI1 and ORAI3 show a differential sensitivity to reactive oxygen species, due to the extracellularly located Cys195 residue which is found in ORAI1, but not in ORAI3. The differential redox sensitivity underlies the differential responses between naïve and T helper lymphocytes, an event that lets T(H) cells

ORAI3, but not ORAI1, was also involved in the activation of PLCδ in response to arachidonic acid, an activation that controls oscillation frequency of Ca2+ spikes triggered by carbachol [92]. ORAI3 channels are overexpressed in estrogen receptor-positive breast cancer cells [93], and it was later demonstrated, using the MCF-7 cancer cell line, that silencing ORAI3 slows down cell cycle and triggers arrest at G1 phase [94]. EGF triggers Ca2+ entry through ORAI3, and the channel is transcriptionally upregulated by the estrogen receptor alpha (ERα) [95]. It is now accepted that cancer cells show a remodeling of ORAI proteins, with an enhanced participation of ORAI3 compared to noncancerous cells, suggesting that heteromerization of

vating TGF-β, Snail and Wnt/β-catenin pathways in prostate cancer cells [87].

transport and tumor growth [85].

12 Calcium and Signal Transduction

**5. ORAI3 and calcium signaling**

proliferate and secrete cytokines in oxidative environments [91].

ORAI3 and ORAI1 is a common feature in malignant transformation [96].

of CREB.

During the past decade, a significant progress was made regarding the molecular description of the proteins involved in store-operated Ca2+ entry. Although some details remain unclear,

**Figure 2.** Activation of SOCE by STIM1/ORAI1 and pathways involved in SOCE-dependent signaling. Panel A: diverse stimuli that triggers the activation of the phospholipase C pathway, such as activation of EGF receptor (EGFR), stimulate the production of inositol 1,4,5-trisphosphate (IP3) which binds and activates IP3 receptor (IP3R) at the endoplasmic reticulum (ER). This activation leads to the release of Ca2+ from the ER, with the subsequent transient depletion of intraluminal [Ca2+] and the activation of STIM1. Ca2+-unbound STIM1 aggregates in oligomers and translocates to plasma membrane (PM)-ER junctions where it binds and activates ORAI1. Extracellular Ca2+ entry through ORAI1 activates multiple Ca2+-dependent targets, as shown in panel B, but also provides a Ca2+ source to replenish intraluminal Ca2+ levels. This replenishment is accomplished by the ER-Ca2+-ATPase which pumps Ca2+ into the ER lumen. Panel B: schematic illustration of the most important pathways regulated by STIM1/ORAI1. AC, adenylyl cyclase; GPCR, G protein-coupled receptor; PM-STIM1, plasma membrane-resident STIM1; p-STIM1, phosphorylated STIM1.

a topology of STIM1-ORAI1 contact sites, selectivity filters in ORAI1, and the role of posttranslational modifications have been reported for both proteins. The involvement of STIM and ORAI proteins in different pathways is now much clearer, and they are now considered master regulators of Ca2+-dependent signaling pathways. However, in many cases pathways were studied in cancer cell lines in vitro, so physiological models are required to evaluate the importance of STIM1 and ORAI1 in the pathophysiology of cells in vivo. Nevertheless, primary cell cultures and established cell lines constitute a widely accepted experimental approach for basic studies in cell signaling and understanding the role of STIM1/ORAI1 in cell biology and cell signaling. With these tools, we have reached the conclusion that STIM1 and ORAI1 are involved in the control of Ca2+ refilling within the ER. More important, STIM1 and ORAI1 directly modulate Ca2+ signaling in a wide variety of pathways, with a significant role in gene expression, cell migration, and tumor cell metastasis (**Figure 2**). Because the expression of STIM1/ORAI1 is deregulated in cancer cells, it is required to evaluate the relative importance of STIM1/ORAI1 as pharmacological targets for the treatment of disease, not only with the use of in vitro cell cultures, but also in animal models for the study of human disease.

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*2.3. Morphology and Mechanical Properties of PLLA-SBA2 Fibres*

PLLA-SBA2 electrospun fibres were sputtered with gold by using a Sputter Coater (Q150T, Quorum Technologies, Darmstadt, Germany) and then inspected by SEM and energy dispersive X-ray spectrometry (EDS) (Auriga 0750, ZEISS, Jena, Germany). Average fibre diameters were calculated by analysing a total of at least 100 fibres for each sample, using the software ImageJ [40]. Regarding mechanical properties, as reported by Ricotti et al. [41], mechanical differences in uniaxial aligned arrays are more pronounced. Thus, aligned fibre mats were used in this paper to investigate more specifically the mechanical properties of PLLA-SBA2 composite fibre mats. Mechanical properties of random and aligned composite fibres mats at room temperature were investigated by uniaxial tensile strength tests using a universal testing machine (K. Frank GmbH, Mannheim, Germany). Each sample was cut into a rectangular shape (with cross-section area of 5 × 4 mm2) using a paper square framework and its thickness is measured by using a digital micrometre (0.02–0.06 mm). Then measurements were carried out at a crosshead speed of 10 mm/min using a 50 N load cell, according to a previous

study [42] and the resulted stress-strain curves are used to obtain Young's modulus, elongation at break and tensile strength.