4. Discussion

Results obtained in the present study are in line with our earlier prediction of CPB development under climate change in the Wielkopolska region, located in the western part of Poland [15]. In that study, two CPB generations were generated following a temperature increase of around 2C for the Wielkopolska region by a model developed on simulation results obtained using meteorological data registered at WinnaGóra, located 60 km south of Poznań, and data obtained after transformation of the recorded data to reflect a temperature increase of 1–6C. Similar results are presented in the present study for Poznań, where two generations of CPB are expected with a probability of 0.95 and 0.99 following a temperature increase of 1.9 and 2.3C, respectively. Comparison of the results of 16 models describing the influence of temperature rises on SGCPBP indicated that thermal conditions in south-western Poland are most similar to those that guarantee the appearance of two generations. On the other hand, an increase in the number of CPB generations in north-eastern Poland required a much higher temperature rise. The main advantage of the current study over the study published in 2013 is not only the increase in the number of localities but also the inclusion of four RCP scenarios, which enable an assessment of regional variations in climate change.

Simulation results produced with the use of RCP scenarios in the present study show that an increase in the number of CPB generations is very likely to appear in Poland. Under three (RCP4.5, RCP6 and RCP8.5) out of the four analyzed scenarios, average SGCPBP calculated for 16 localities is going to exceed 75% after 2039. The only scenario which shows that, at the end of the century, the situation will not differ very much from that observed in 1986–2005 is RCP2.6. However, even under that scenario, simulations showed a shortening of the CPB first generation development time, especially in the northern (Gdańsk), north-eastern (Olsztyn, Białystok), eastern (Lublin) and southern parts of central Poland (Kielce). The same regions were indicated as being the most vulnerable to shortening of the CPB first generation development from simulation results under scenarios RCP4.5, RCP6 and RCP8.5. Moreover, analysis of the pest development under scenarios RCP4.5, RCP6 and RCP8.5 showed that the second generation usually developed faster than the first generation. We decided not to compare the development time of the generations obtained in simulations on real data and under scenario RCP2.6 because of the excessive number of SGCPBP results lower than 50%.

generated the appearance of the second pest generation at Lublin and Białystok, respectively. At Kielce and Olsztyn, temperature rises of 2.5 and 3C were needed to trigger the appearance of the second generation, whereas at Gdańsk, the probability of the appearance of the second

With a probability of 99%, a temperature rise of 1.4C generated the second generation of CPB at Opole. At Katowice and Rzeszów, temperature had to increase by 1.9C. At Kraków, Szczecin and Wrocław, a 2C temperature rise triggered the appearance of the pest second generation. At ZielonaGóra and Łódź temperature had to rise by 2.2C. A temperature increase of 2.3, 2.4 and 2.5C generated the appearance of the second pest generation at Poznań, Toruń and Lublin. At Kielce and Białystok, temperature rises of 2.9 and 3.2C were needed to trigger the appearance of the second generation, whereas for Gdańsk and Olsztyn, the models did not generate the appearance of the second generation with a probability of 99%.

Results obtained in the present study are in line with our earlier prediction of CPB development under climate change in the Wielkopolska region, located in the western part of Poland [15]. In that study, two CPB generations were generated following a temperature increase of around 2C for the Wielkopolska region by a model developed on simulation results obtained using meteorological data registered at WinnaGóra, located 60 km south of Poznań, and data obtained after transformation of the recorded data to reflect a temperature increase of 1–6C. Similar results are presented in the present study for Poznań, where two generations of CPB are expected with a probability of 0.95 and 0.99 following a temperature increase of 1.9 and 2.3C, respectively. Comparison of the results of 16 models describing the influence of temperature rises on SGCPBP indicated that thermal conditions in south-western Poland are most similar to those that guarantee the appearance of two generations. On the other hand, an increase in the number of CPB generations in north-eastern Poland required a much higher temperature rise. The main advantage of the current study over the study published in 2013 is not only the increase in the number of localities but also the inclusion of four RCP scenarios,

Simulation results produced with the use of RCP scenarios in the present study show that an increase in the number of CPB generations is very likely to appear in Poland. Under three (RCP4.5, RCP6 and RCP8.5) out of the four analyzed scenarios, average SGCPBP calculated for 16 localities is going to exceed 75% after 2039. The only scenario which shows that, at the end of the century, the situation will not differ very much from that observed in 1986–2005 is RCP2.6. However, even under that scenario, simulations showed a shortening of the CPB first generation development time, especially in the northern (Gdańsk), north-eastern (Olsztyn, Białystok), eastern (Lublin) and southern parts of central Poland (Kielce). The same regions were indicated as being the most vulnerable to shortening of the CPB first generation development from simulation results under scenarios RCP4.5, RCP6 and RCP8.5. Moreover, analysis

which enable an assessment of regional variations in climate change.

generation did not exceed 93%.

76 Potato - From Incas to All Over the World

4. Discussion

As expected, the lowest values for SGCPBP were generated under scenario RCP2.6, whereas the highest were noted under RCP8.5. The SGCPBP values produced by the former are usually lower than 75%. That is why we did not use it to specify the regions to be threatened by CPB in the future. The values for SGCPBP produced by RCP8.5 are usually higher than 75%, but there are almost no differences in this analyzed parameter between localities, so this scenario was also not used for specification of the regions at most risk from CPB in the future. On the other hand, scenarios RCP4.5 and RCP6 can be helpful in identifying regions at risk from CPB in the future. Both produced quite differential results, usually higher than 75%. Comparison of simulation results obtained under scenario RCP4.5 enables identification of the south-western region (Opole, Wrocław), the south-eastern (Rzeszów), eastern (Lublin) and southern parts of central Poland (Kielce), as being the most threatened by CPB in the future. Simulations under scenario RCP6.0 additionally included the south of Poland (Katowice) as one of the region most at risk of CPB.

Comparison of SGCPBP obtained in simulations on real and transformed data also enables identification of the regions vulnerable to higher changes in SGCPBP. Based on scenarios RCP2.6, RCP4.5 and RCP6.0, the south of central Poland (Kielce) and the eastern part of Poland (Lublin) should be included into that category. The predicted increase in SGCPBP obtained in simulations under these scenarios for these two localities distinctly differs from the rest. According to simulation results from scenario RCP8.5, it appears that besides these two localities, another three (Białystok, Gdańsk and Olsztyn) are more vulnerable to increase in SGCPBP than other localities.

Considering the results of the study dealing with the risk of CPB third generation appearance, it seems that the south-western region (Opole), eastern region (Lublin) and southern part of central Poland (Kielce) may face the most problems caused by increased numbers of CPB generations.

Results obtained in the present study are also in line with predictions of CPB development under expected climate change in the Czech Republic presented by Kocmankowa et al. [28], who used a simulation performed with the use of the CLIMEX model to show a growing danger of an increase in the number of CPB generations. This is in line with the predictions by Menéndez [29], Das et al. [30] and Sangle et al. [31], who expected greater numbers of generation of so-called "stop and go" insects following climate change. Kocmankowa et al. [28] also predicted a widening of the area of CPB occurrence and a shifting of the pest to higher altitudes. The significant increase in SGCPBP in the Małopolska upland located in the southern part of central Poland (Kielce) showed in our study confirms the findings of Kocmankowa et al. [28]. Similar results were also presented by Pulatov et al. [32], who analyzed the effect of climate change on the potential spread of the Colorado potato beetle in Scandinavia. They showed a substantial increase in the frequency of years in which the temperature requirement for development of one generation was fulfilled. Additionally, they indicated regions where two generations per year may occur more often.

The results of the simulations performed by Žalud et al. [33] for middle Europe, including Poland, also show an increase in the number of CPB generations per year based on the temperature increase predicted by various scenarios. Possible increases in the number of CPB generations are also expected in some areas of Russia by Popova [34], who used cartographic modeling to show that this phenomenon is caused by an increase in the sum of effective air temperatures in those territories.

References

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10.1016/j.pestbp.2015.08.002

the USSR. ZaschitaRastenii. 1978;6:44-45

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[1] Li C, Liu H, Huang F, Cheng DF, Wang JJ, Zhang YH, Sun JR, Guo WC. Effect of temperature on the occurrence and distribution of colorado potato beetle (Coleoptera: Chrysomelidae) in

Simulations of Colorado Potato Beetle Development in Poland Based on Four Climate Change Scenarios

http://dx.doi.org/10.5772/intechopen.70777

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[2] Ferro DN, Logan JA, Voss RH, Elkinton JS. Colorado potato beetle (Coleoptera: Chrysomelidae) temperature-dependent growth and feeding rates. Environmental Entomology.

[3] Hsiao, T. H. 1985. Ecophysiological and genetic aspects of geographic variations of the Colorado potato beetle, p. 63-77. In D. N. Ferro and R. H. Voss (ed.), Proceedings of Symposium on the Colorado Potato Beetle, XVIIth International Congress of Entomology. Agricultural Experiment Station Research Bulletin 704. University of Massachusetts, Amhers

[4] Jolivet P. The Colorado beetle menaces Asia (Leptinotarsa decemlineata Say) (Coleoptera:

[5] Malekmohammadi M., Galehdari H. Target site insensitivity mutations in the AChE 15 enzyme confer resistance to organophosphorous insecticidesin Leptinotarsa decemlineata (SAY), Pesticide Bio16 chemistry and Physiology. 2015; 126:85-91. DOI: http://dx.doi.org/

[6] Vlasova VA. A prediction of the distribution of Colorado beetle in the Asiatic territory of

[7] Worner SP. Ecoclimatic assessment of potential establishment of exotic pests. Journal of

[8] Węgorek W. The Colorado potato beetle (Leptinotarsa decemlineata Say). Prace Naukowe

[9] Kałmuk J., Pawłowski J: Księga gatunków obcych inwazyjnych w faunie Polski, 2008. Available from: http://www.iop.krakow.pl/gatunkiobce/default6db7.html?nazwa=opis&

[10] Mrówczyński M. Występowanie i zwalczanie stonki ziemniaczanej (Leptinotarsa decemlineata Say) w latach 1978–1999. Progress in Plant Protection/Postępy w Ochronie Roślin. 2000;40

[11] Pawińska M, Mrówczyński M. Występowanie i zwalczanie stonkiziemniaczanej (Leptinotarsa decemlineata Say) w latach 1978–1999. Progress in Plant Protection/Postępy w

[12] Węgorek P, Stachecki S. Ryzyko wystąpienia odporności agrofagów na środki ochrony roślin. In: Proceedings of the Conference: Integrowana ochrona ziemniaka przed choro-

[13] Wójtowicz A, Jörg E. Prognozowanie rozwoju stonki ziemniaczanej za pomocą program ukomputerowego SIMLEP. Progress in Plant Protection/Postępy w Ochronie Roślin.

bami, szkodnikami i chwastami; Poland Poznań. September 1999. p. 83-92

Chrysomelidae). L'Entomologiste. 1991;47:29-48

China. Environmental Entomology. 2014;43:511-519. DOI: 10.1603/EN13317

The differences in the effect of climate change on CPB development between regions shown in the present study are also in consistent with the findings of Wittchen and Freyer [35], who analyzed the impact of temperature increases at two localities in Germany (Potsdam and Ulm) on the appearance of subsequent developmental stages of CPB. Simulations performed under real temperatures generated faster development of CPB first generation at Potsdam (30 m a. s. l.) than at Ulm (470 m a. s. l.) (by 5 days). But, under increased temperatures, CPB developed faster at Ulm than at Potsdam (by 3 days). A similar situation was noticed while comparing the development of CPB in Poland at Toruń (46 m. a. s. l.) and Kielce (270 m. a. s. l.). Using real data, development of CPB first generation at Toruń was 5 days shorter than at Kielce, while under transformed data and depending on the RCP scenario at Toruń development was 2–3 days longer than at Kielce.

The results of this study clearly indicate not only increased rates of CPB development following expected climate change across Poland, but also the regions exposed to the most rapid changes in the number of pest generations. But, one has to be aware that the interaction among the environmental factors is very complex and changeable. Most insect pests can adapt to a wide range of environments through selection and evolution. Therefore, prediction based on factor-limited simulations produce results with limited accuracy. On the other hand, only simulations can aid a rapid investigation of the effects of a change in a real life situation that will take place in the future over several years. From simulation results, a problem expected in the future can be mitigated now. So, systematic monitoring of potato crops in the regions indicated in the present study as the most threatened by the appearance of additional generations of CPB should be our first priority. Knowledge about pest trends gained in simulations coupled with results of field monitoring allows the determination of the feasibility of using certain pest management strategies.


**2. The fuel cells' principle of work**

The main components of a fuel cell are two electrodes isolated from each other with a membrane (either solid or liquid electrolyte). The added parts are usually to improve the functioning of the cell. According to Eq. (1) [5], the gas fuel (hydrogen) oxidized at the anode to produces electrons and cations during entering of the oxidant from the cathode. These released electrons depart the anode through an external circuit and create an electric current before reaching the cathode. The electrons current can be utilized in working any load [5].

$$2H\_2 \rightarrow 4H^+ + 4e^- \tag{1}$$

The positive hydrogen ions diffuse through the membrane and recombine with the exhausted electrons that reached at the cathode to reproduce H2 gas. Finally, the produced hydrogen atoms combine with the oxygen atoms to generate water as the only waste of the complete reaction. Eqs. (2) and (3) show the reduction of oxygen and the fuel cell reaction, respectively [6, 7].

$$2\text{ O}\_2 + 4\text{ H}^+ + 4e^- \rightarrow 2\text{ H}\_2\text{O}\tag{2}$$

$$2H\_2 + O\_2 \rightarrow 2H\_2O \text{ + heat} \tag{3}$$

**229**

**3.1 The end plates**

**Figure 2.**

**3.2 The bipolar plates**

*Fuel Cells as a Source of Green Energy*

*DOI: http://dx.doi.org/10.5772/intechopen.89736*

of the fuel cell is displayed in **Figure 1**.

*A scheme of oxidization processes at the electrodes [13].*

the oxidization reaction schematically.

**3. The main components of fuel cells**

connection. These parts are shown in **Figure 3**.

through it in one direction only which is the direction of the cathode [8]. To reduce the reaction energy on the two electrodes, a catalyst is added [9, 10]. The structure

Theoretically, the anodic substance could be any material that gives up electrons such as hydrogen, methane, kerosene, or even carbon. But among all, hydrogen is preferred due to its availability, has a high energy density, and good reactivity with catalysts [12]. Also, the best chose of oxidant agent is oxygen gas because it is easily subjected to a reduction reaction and is much available in the air. **Figure 2** illustrates

In addition to the two electrodes and their separator electrolyte, other additional parts are required for specific tasks such as gases flow and electrical insulation or

The outer sides of the anode and the cathode are supported mechanically by plastic plates which are called the endplates. Their main jobs are to save the fuel cell

A fuel cell has two bipolar plates each one consists of a current collector and separator plate. They are manufactured from a metal or a carbon-filled composite (conductive polymer) [16]. Researchers suggested several designs of

component and to prevent the reacting gasses from leaking [15].

Fuel cell' efficiency is greatly influenced by the quality of the electrolyte. It increases with increasing the membrane ability to block electrons generated from the oxidation of the anode from reaching the cathode. Therefore, the electrolyte must be chosen with caution as it acts as a filter that allows only positive ions to pass

**Figure 1.** *The components of fuel cell [11].*

*Fuel Cells as a Source of Green Energy DOI: http://dx.doi.org/10.5772/intechopen.89736*

*Thermodynamics and Energy Engineering*

**2. The fuel cells' principle of work**

electrons current can be utilized in working any load [5].

and the fuel cell reaction, respectively [6, 7].

Fuel cells are zero pollution' power generators. They work as an electrochemical converter to chemically produce electrical energy from gas fuels. The conversion occurs directly by the chemical reactions and without burning of the fuel. Even better, they are not following the thermodynamic laws that limit most of the power plants. This chapter presents the operation principle of fuel cells, their main components, and finally the newer trends in developing these cells for commercial uses.

The main components of a fuel cell are two electrodes isolated from each other with a membrane (either solid or liquid electrolyte). The added parts are usually to improve the functioning of the cell. According to Eq. (1) [5], the gas fuel (hydrogen) oxidized at the anode to produces electrons and cations during entering of the oxidant from the cathode. These released electrons depart the anode through an external circuit and create an electric current before reaching the cathode. The

2 *H*2 → 4 *H*+ + 4 *e*

*O*2 + 4 *H*+ + 4 *e*

The positive hydrogen ions diffuse through the membrane and recombine with the exhausted electrons that reached at the cathode to reproduce H2 gas. Finally, the produced hydrogen atoms combine with the oxygen atoms to generate water as the only waste of the complete reaction. Eqs. (2) and (3) show the reduction of oxygen

−

Fuel cell' efficiency is greatly influenced by the quality of the electrolyte. It increases with increasing the membrane ability to block electrons generated from the oxidation of the anode from reaching the cathode. Therefore, the electrolyte must be chosen with caution as it acts as a filter that allows only positive ions to pass

− (1)

→ 2 *H*2*O* (2)

2 *H*2 + *O*2 → 2 *H*2*O* + *heat* (3)

**228**

**Figure 1.**

*The components of fuel cell [11].*

**Figure 2.** *A scheme of oxidization processes at the electrodes [13].*

through it in one direction only which is the direction of the cathode [8]. To reduce the reaction energy on the two electrodes, a catalyst is added [9, 10]. The structure of the fuel cell is displayed in **Figure 1**.

Theoretically, the anodic substance could be any material that gives up electrons such as hydrogen, methane, kerosene, or even carbon. But among all, hydrogen is preferred due to its availability, has a high energy density, and good reactivity with catalysts [12]. Also, the best chose of oxidant agent is oxygen gas because it is easily subjected to a reduction reaction and is much available in the air. **Figure 2** illustrates the oxidization reaction schematically.


**5. G-quad in luminescence-based sensors**

Although applied widely, fluorescent labelling for sensor development has some limitations. Labelling the oligonucleotide covalently can reduce the binding affinity or selectivity of the oligonucleotide, which in turn hampers the efficiency of the assay. Besides, fluorescent labelling is costly and time consuming. Therefore, the use of luminescent probes in sensor development was an attractive solution. Unlike fluorescent probes, luminescent probes are not covalently attached to the nucleic acid; instead, it can bind with DNA through end stacking or electrostatic interactions, intercalation and groove binding. Such interactions will not affect the functionality of the DNA [43].

A label-free G-quad assay for metal detection was developed by Ma et al. This assay was based on the transition of a DNA structure, which involved specially designed oligonucleotide that is rich in guanine and cytosine. The transition of DNA structure would be from a quadruplex to duplex conformation induced by silver (Ag+ ) ions. In this assay, platinum (II) metallo intercalator was used for the switch on/ switch off response towards the Ag+ ions. Initially, a low luminescence background was generated due to the weak interaction of platinum (II) metallo intercalator and the G-quad structure without the Ag+ ions. However, after adding the Ag+ ions, cystosine-Ag+ -cytosine mismatched base pairing occurred, causing the G-quad structure to revert to a duplex structure. This allowed the intercalation of the platinum(II) metallo intercalator to the duplex DNA structure and generates a luminescent signal [44].

to dissociate the G-quad structure, the strong Pb2+ chelator DOTA (1,4,7,10-tetraazacyclodo‐ decane-1,4,7,10-tetraacetic acid) was used as it has high binding capacity against Pb2+ to remove Pb2+ from the G-quad structure, leaving the G-quad structure to dissociate and form a DNA

As reported by Kankia and co-workers, a further improvement to their original QPA method was reported [42]. The improved method involves the use of two linear processes that are the QPA and linear nicking amplification, which is based on the study done by Galas and coworkers. The probe DNA was hybridized with the target DNA and polymerase was used to extend the target DNA strand and form QPA-PBS (primer-binding site). Then, the nicking enzyme Nt.BSTNBI was introduced to nick the target strand and release the QPA-primerbinding strand. The QPA-primer-binding strand will bind to the QPA primer and the poly‐ merase will function to extend the strand further. The primer will then dissociate to emit fluorescence signals. The following priming step of QPA will be initiated again when the next primer binds with the QPA-primer binding strand. This method was reported to be able to

reduce the background activity to allow sufficient sensitivity of the assay (Figure 5).

Although applied widely, fluorescent labelling for sensor development has some limitations. Labelling the oligonucleotide covalently can reduce the binding affinity or selectivity of the oligonucleotide, which in turn hampers the efficiency of the assay. Besides, fluorescent labelling is costly and time consuming. Therefore, the use of luminescent probes in sensor development was an attractive solution. Unlike fluorescent probes, luminescent probes are not covalently attached to the nucleic acid; instead, it can bind with DNA through end stacking or electrostatic interactions, intercalation and groove binding. Such interactions will not affect

duplex with the complementary strand again [41].

128 Nucleic Acids - From Basic Aspects to Laboratory Tools

**Figure 5.** Principle of the QPA integrating nicking enzyme for diagnostics.

**5. G-quad in luminescence-based sensors**

the functionality of the DNA [43].

Besides metal detection assays, the same group also developed an assay for the detection of gene deletion (Figure 6). They designed a split G-quad assay whereby it consists of two short oligonucleotides, namely P1 and P2. Both of these strands have complementary regions that can recognize the deletion site of the chemokine receptor gene CCR5 and also contain guaninerich overhangs. These guanine-rich overhangs of both strands can form a split G-quad in close proximity. CCR5 was used as the target because it was reported to be a co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strain and it allows HIV-1 to enter the CD4+ T cells. Mutation on CCR5 gene caused no expression of functional protein, which gives rise to the resistance against HIV infection. In this assay, P1 and P2 strands were able to hybridize with the wild-type CCR5 DNA strand. Due to the large spatial separation between both G-rich overhangs of P1 and P2, a split G-quad structure was not formed, causing the generation of a lower luminescent signal of the iridium(III) complex. However, mutant of the CCR5 DNA sequence that is shorter in length can cause the two G-quad-forming sequences of P1 and P2 to close proximity upon hybridizing with the shorter mutant sequence. This resulted in the formation of the split G-quad, generating a switch-on luminescence effect of the iridium (III) complex [45].

**Figure 6.** Split G-quad luminescent turn-on detection for the detection of gene deletion of HIV DNA sequence.


**3. Stress evaluation procedure and results**

The in-situ stress measurements were conducted under the following situations:

**Figure 12.** *Shut-in pressure related to Hydraulic fracturing.*

#### **Figure 13.** *Experiment 4.*

Topography is the study of the land surface and forms the basis for landscapes. For example, topography refers to mountains, valleys, rivers, and craters on earth surface. If a tunnel is being excavated beneath a land consisting of different rock covers or overburden layers, anisotropic conditions are imminent [18].

#### **3.1 Fracture orientation analysis—PLANE**

The orientation (strike, dip angle and dip direction) of induced fracture traces obtained from impression packer testing is determined with the program PLANE in consideration of the borehole diameter and orientation [17]. Also, it differs with fracture traces as shown below.

**Case I:** Vertical borehole—angle from north to mark (0–360 degrees)

**Case Ia:** Fracture traces parallel to the borehole axis. Distance from mark (reference line) to fracture trace (**Figure 14**).

**Case Ib:** Inclined fractures (**Figure 15**).

**Case II:** Inclined borehole—angle from vertical line to reference mark (0–360 degrees)

*Hydraulic Fracturing in Porous and Fractured Rocks DOI: http://dx.doi.org/10.5772/intechopen.106552*

**Figure 14.** *Trace of the fracture is parallel to the mark found both sides.*

**Figure 15.** *Inclined fractures.*

**Case IIa:** Fracture traces parallel to the borehole axis. **Case IIb:** Inclined fractures

In all cases the result of the calculations is the strike direction (North Over East), the direction of inclination (North over East) and the inclination (90 degrees = vertical) of the fracture plane.


**Abstract**

Anaerobic digestion is a widely used process for waste treatment and energy production. This natural process takes place in a controlled environment, anaerobic digesters. Mixing is one of the main operating parameters. The understanding of the flows during the agitation of the medium is crucial for the optimization of the process yield. In fact, the mass and heat transfers are enhanced by the agitation. However, the complex biochemical reactions can be inhibited with overly vigorous agitation. A detailed and in-depth understanding of the phenomena occurring during agitation requires modeling studies. In this chapter, we propose a general approach, based on computational fluid mechanics (CFD), to analyze the mechanical mixing of an anaerobic reactor. We apply this work to the anaerobic digestion of the sugarcane vinasse, which is a liquid waste generated during the production of alcohol. The single-phase Reynolds-averaged Navier-Stokes (RANS) simulations of mechanical agitation of Newtonian fluids for different rotational speeds are presented. The equations system is closed with the standard k-epsilon turbulence model. The flow field is analyzed with the velocity profiles, the Q and Lambda2 fields, the pressure and the vorticity.

**Keywords:** anaerobic digestion, computational fluid dynamics (CFD), Newtonian, mechanical mixing, vinasse, RANS


**4. Intracranial compliance in real world**

The next paragraphs will provide information on how to incorporate waveform information of ICP into clinical daily life, adding this information to the clinical set and adjunct with other diagnostic methods in different pathologies. It is worth mentioning that this information has been shown to be useful in situations where subjects present suspicion, risk or diagnosis of changes in ICC.

#### **4.1 Aneurysm**

Non-traumatic subarachnoid hemorrhage (SAH) is a situation that often results from the rupture of an intracerebral aneurysm [25]. SAH is associated with high morbidity and mortality and requires a multidisciplinary treatment, because of its high risk of complications [26].

Upon recognition, improved outcomes are dependent upon treatment by qualified high-volume centers with adequate neurovascular teams. Expeditiously determining the precipitating factor and subsequent mitigation of the cause(s) are the initial primary focus. Treatment involves early securing of a ruptured aneurysm, whether a surgical procedure or endovascular. Prior to securing the aneurysm, securing the airway, maintaining proper circulation, treating hydrocephalus, and managing blood pressure remain top priorities. After intervention, ICU observation and routine exams are compulsory.

Once patients presenting with aneurysmal SAH are acutely stabilized, they are evaluated for pathology-specific complications such as development of hydrocephalus and re-hemorrhage. Various grading scales are employed early in management to communicate the severity and prognosis of the pathology. Following stabilization and initial evaluation, patients should be transferred and admitted to intensive care units with a multidisciplinary team. Interim/short-term acute care strategies are employed to prevent rebleeding, assess hydrocephalus, maintain normotension, and reverse anticoagulant/antiplatelet agents. The risk of acute rebleed and long-term prevention of rebleed is not completely attenuated until aneurysm exclusion is performed.

Concurrent to the those risks above mentioned, in the extreme acute phase (first 48 hours) of SAH, the encephalic microvascular constriction promoted by hemoglobin degradation in the subarachnoid space may lead to a low cerebral blood flow (CBF) phenomena, with potential for brain swelling and ICC impairment. Techniques for monitoring ICC and CBF (such as transcranial Doppler) may play a crucial role in this phase.

Later, in the subsequent SAH phase, an inverse behavior is commonly seen in accordance with bleed severity, the so-called hyperemic phase. In this situation subjects present microvascular dilation, this time leading to ICC impairment for excess of CBF. An optimal therapy here is adapting CBF for satisfactory neuronal supply, under ICC adequate limits.

An additional threat for patients in this phase is the development of vasospasm, a complication which elevates risk of delayed cerebral ischemia, in opportunities needing endovascular management. The latter, associated with medical complications including fever, hyperglycemia, hyponatremia, cardiac and pulmonary complications, deep venous thrombosis and anemia may raise risk of ICC impairment. While scores classifications exist to determine an admission grade in order to provide prognostic information, outcomes are influenced by many additional

**7**

*Intracranial Pressure Waveform: History, Fundamentals and Applications in Brain Injuries*

items, including a patient's values and preferences, comorbidities, social support,

The incidence and survival of patients with neuro-oncologic conditions have been increasing. Both primary central nervous and other types of cancer patients live longer due to early diagnosis and better treatment options. Global Burden Disease Study in 2016, there were 330,000 incident cases of CNS cancer and 227,000 deaths worldwide that year. It reflects the 17.3% increase in incidence

Extension of life expectancy and on the incidence of cancer itself predisposes to an increment in the occurrence of a variety of neurologic complications that can

These conditions often result in hospital admissions, generally in an ICU bed, creating a heavy burden to the health care system since primary cancer patients' treatment costs 20-times more than age-matched controls without cancer [33].

The complications could occur due to a direct result of the tumor itself, to an indirect effect of cancer, or as a result of chemotherapy, radiotherapy, and other medical interventions. Recognizing the mechanism might help one early diagnosis and initiate treatment. As a mass effect directly, or even a compromise of CSF transit because of ventricle compression, intracranial neoplasm may lead to ICC impairment.

The World Health Organization considers traumatic brain injury (TBI) an important global health priority as it is a critical public health problem involving young adults worldwide. The leading causes of TBI are road traffic collisions, falls and interpersonal violence. This injury not only causes a large number of deaths, impairments and disabilities for individuals and their families, but also incurs great economic cost to healthcare systems due to required long-term care, rehabilitation,

TBI can be classified by clinical severity (mild, moderate, or severe) according to the Glasgow Coma Scale (GCS); pathoanatomic type (focal or diffuse) according to the extent of damaged area; and mechanism of injury (penetrating or blunt)

The TBI-related cellular injury involves two different processes. The primary damage occurs on the moment of trauma, immediately by the direct impact and/or structural lesion. It includes vascular and tissue tearing that causes various types of hemorrhage and nerve fibers disruption (axotomy). The secondary damage involves cellular reactive processes such as inflammation and biochemical cascades that gradually develop over the course of hours, days, even weeks after the trauma. It causes metabolic changes potentially leading to brain swelling or hydrocephalus but can also be caused by low blood pressure, hypoxia, seizures, or central nervous system infection [37, 38].

Both processes are intertwined and can contribute to complications, for instance, hemorrhagic progression of a contusion, a breakdown in the blood–brain barrier (BBB), and increased intracranial pressure (ICP). The expansion of an intracranial bleeding not only alters the dynamic shared space of encephalic parenchyma, vascular structures, and cerebral spinal fluid (CSF) inside cranial cavity – inferred intracranial compliance – but also triggers cytotoxic responses of brain cells. In addition, if there is a dysfunction of BBB its permeability changes letting plasma, proteins and proinflammatory mediators influx into the interstitial compartment causing edema, neurotransmitters imbalance, compressing all structures [38, 39].

*DOI: http://dx.doi.org/10.5772/intechopen.94077*

resilience, and time for recovery [27–31].

result in high morbidity and mortality [32, 33].

**4.2 Tumor**

between 1990 and 2016.

**4.3 Traumatic brain injury**

and loss of productivity [34].

according to the kinematics (**Table 1**) [35–38].

*Intracranial Pressure Waveform: History, Fundamentals and Applications in Brain Injuries DOI: http://dx.doi.org/10.5772/intechopen.94077*

items, including a patient's values and preferences, comorbidities, social support, resilience, and time for recovery [27–31].

### **4.2 Tumor**

*Advancement and New Understanding in Brain Injury*

**4. Intracranial compliance in real world**

**4.1 Aneurysm**

high risk of complications [26].

crucial role in this phase.

supply, under ICC adequate limits.

subjects present suspicion, risk or diagnosis of changes in ICC.

ICU observation and routine exams are compulsory.

indicators of diminished compliance and can also be present in patients with normal ICP [24]. Finally, C waves are products of cardiac and respiratory cycles interactions.

The next paragraphs will provide information on how to incorporate waveform information of ICP into clinical daily life, adding this information to the clinical set and adjunct with other diagnostic methods in different pathologies. It is worth mentioning that this information has been shown to be useful in situations where

Non-traumatic subarachnoid hemorrhage (SAH) is a situation that often results from the rupture of an intracerebral aneurysm [25]. SAH is associated with high morbidity and mortality and requires a multidisciplinary treatment, because of its

Upon recognition, improved outcomes are dependent upon treatment by qualified high-volume centers with adequate neurovascular teams. Expeditiously determining the precipitating factor and subsequent mitigation of the cause(s) are the initial primary focus. Treatment involves early securing of a ruptured aneurysm, whether a surgical procedure or endovascular. Prior to securing the aneurysm, securing the airway, maintaining proper circulation, treating hydrocephalus, and managing blood pressure remain top priorities. After intervention,

Once patients presenting with aneurysmal SAH are acutely stabilized, they are evaluated for pathology-specific complications such as development of hydrocephalus and re-hemorrhage. Various grading scales are employed early in management to communicate the severity and prognosis of the pathology. Following stabilization and initial evaluation, patients should be transferred and admitted to intensive care units with a multidisciplinary team. Interim/short-term acute care strategies are employed to prevent rebleeding, assess hydrocephalus, maintain normotension, and reverse anticoagulant/antiplatelet agents. The risk of acute rebleed and long-term prevention of rebleed is not completely attenuated until aneurysm exclusion is performed. Concurrent to the those risks above mentioned, in the extreme acute phase (first 48 hours) of SAH, the encephalic microvascular constriction promoted by hemoglobin degradation in the subarachnoid space may lead to a low cerebral blood flow (CBF) phenomena, with potential for brain swelling and ICC impairment. Techniques for monitoring ICC and CBF (such as transcranial Doppler) may play a

Later, in the subsequent SAH phase, an inverse behavior is commonly seen in accordance with bleed severity, the so-called hyperemic phase. In this situation subjects present microvascular dilation, this time leading to ICC impairment for excess of CBF. An optimal therapy here is adapting CBF for satisfactory neuronal

An additional threat for patients in this phase is the development of vasospasm, a complication which elevates risk of delayed cerebral ischemia, in opportunities needing endovascular management. The latter, associated with medical complications including fever, hyperglycemia, hyponatremia, cardiac and pulmonary complications, deep venous thrombosis and anemia may raise risk of ICC impairment. While scores classifications exist to determine an admission grade in order to provide prognostic information, outcomes are influenced by many additional

**6**

The incidence and survival of patients with neuro-oncologic conditions have been increasing. Both primary central nervous and other types of cancer patients live longer due to early diagnosis and better treatment options. Global Burden Disease Study in 2016, there were 330,000 incident cases of CNS cancer and 227,000 deaths worldwide that year. It reflects the 17.3% increase in incidence between 1990 and 2016.

Extension of life expectancy and on the incidence of cancer itself predisposes to an increment in the occurrence of a variety of neurologic complications that can result in high morbidity and mortality [32, 33].

These conditions often result in hospital admissions, generally in an ICU bed, creating a heavy burden to the health care system since primary cancer patients' treatment costs 20-times more than age-matched controls without cancer [33].

The complications could occur due to a direct result of the tumor itself, to an indirect effect of cancer, or as a result of chemotherapy, radiotherapy, and other medical interventions. Recognizing the mechanism might help one early diagnosis and initiate treatment. As a mass effect directly, or even a compromise of CSF transit because of ventricle compression, intracranial neoplasm may lead to ICC impairment.

#### **4.3 Traumatic brain injury**

The World Health Organization considers traumatic brain injury (TBI) an important global health priority as it is a critical public health problem involving young adults worldwide. The leading causes of TBI are road traffic collisions, falls and interpersonal violence. This injury not only causes a large number of deaths, impairments and disabilities for individuals and their families, but also incurs great economic cost to healthcare systems due to required long-term care, rehabilitation, and loss of productivity [34].

TBI can be classified by clinical severity (mild, moderate, or severe) according to the Glasgow Coma Scale (GCS); pathoanatomic type (focal or diffuse) according to the extent of damaged area; and mechanism of injury (penetrating or blunt) according to the kinematics (**Table 1**) [35–38].

The TBI-related cellular injury involves two different processes. The primary damage occurs on the moment of trauma, immediately by the direct impact and/or structural lesion. It includes vascular and tissue tearing that causes various types of hemorrhage and nerve fibers disruption (axotomy). The secondary damage involves cellular reactive processes such as inflammation and biochemical cascades that gradually develop over the course of hours, days, even weeks after the trauma. It causes metabolic changes potentially leading to brain swelling or hydrocephalus but can also be caused by low blood pressure, hypoxia, seizures, or central nervous system infection [37, 38].

Both processes are intertwined and can contribute to complications, for instance, hemorrhagic progression of a contusion, a breakdown in the blood–brain barrier (BBB), and increased intracranial pressure (ICP). The expansion of an intracranial bleeding not only alters the dynamic shared space of encephalic parenchyma, vascular structures, and cerebral spinal fluid (CSF) inside cranial cavity – inferred intracranial compliance – but also triggers cytotoxic responses of brain cells. In addition, if there is a dysfunction of BBB its permeability changes letting plasma, proteins and proinflammatory mediators influx into the interstitial compartment causing edema, neurotransmitters imbalance, compressing all structures [38, 39].

#### **Table 1.**

*Different classifications of TBI.*

As a result of this intricate association the ICP may rise if intrinsic compensatory mechanisms are not preserved and the sustained hypertension can prevent adequate perfusion depriving the brain of oxygen and nutrition. The combination of all situations related to TBI mentioned above need specific and adequate management through the whole trauma assistance from the pre-hospital setting to the critical care unit (CCU) and subsequent rehabilitation [36, 40].

The main method of assessment and management of severe TBI is monitoring and treatment of ICP. It is a level II-B of evidence recommended to reduce inhospital and 2-week post-injury mortality [23, 41].

Neurocritical care specialists routinely base their clinical reasoning looking at the absolute value of ICP – measured in mmHg or cmH2O – combined with imaging exams – CT-scan or MRI. However, the numbers may not translate the entire complexity of intracranial dynamic. It is suggested that the ICP waves and the study of its morphology could bring differential evidence of altered intracranial compliance and changes of pressure regimen [14, 23, 42, 43].

A qualitative analysis of the ICP waveform [44] described the relationship between amplitude of ICP pulse wave, values of ICP, values of cerebral perfusion pressure (CPP), and the outcome of severe head-injured patients. Intracranial hypertension was evidenced by absolute values of ICP and CT-scan parameters. In those with fatal outcomes there was an increase in the ICP waveform amplitude along with an increase of ICP value up to 25 mmHg, however, above this value the amplitude began to decrease. This breakpoint trend in the amplitude-value relationship was not present in patients with good/moderate outcome. Thus, it is suggested that the physics involving ICP, CPP and parenchyma dynamics inside intracranial cavities was somehow translated into the waveforms, and its analysis and correlations could be a useful additional tool for outcome prediction.

 Plateau waves are associated with working cerebrovascular reactivity and occur in situations of

**9**

**Figure 5.**

**Figure 6.**

*Intracranial Pressure Waveform: History, Fundamentals and Applications in Brain Injuries*

decreased volume-pressure compensatory reserve such as TBI and many other brain pathologies. It consists of sudden increases in ICP to peaks of 40-100 mmHg that persists for 5–20 minutes [14, 45, 46]. The study observed plateau waves in 44% of the patients and that abrupt increase of ICP above 40 mmHg was associated with an increase in amplitude of ICP pulse waveform and also with important decrease in CPP, cerebral blood flow and oxygenation, despite stable cardiovascular variables

When analyzing ICP pulse waveform during plateau waves, a statistically significant increase in amplitude and a change in its shape were noted. The ICP pulse

Although slight increases in ICP that last for short period are not usually associated with poor outcome, if plateau waves are sustained over 30 minutes it could have a negative impact on patient's recovery as intracranial hypertension compromises cerebral perfusion and implicates neuronal deterioration [14, 23, 42, 43, 46]. The analysis of ICP absolute values and waveform patterns over time could provide important information for early detection of ICH in TBI patients of

The debate about how ICP waveform analysis could provide improved clinical benefit and a more actionable evidence to bedside addresses integrated metrics on brain's intrinsic compensatory capacity (autoregulation) and oxygenation, besides computational analysis of multiple continuous streams of neuro-monitoring data and equipment development to easily display this information [14, 23, 42–46]. In this way, non-invasive techniques are coming forward to give quick ICP information to neurocritical care team, including transcranial Doppler, optic nerve sheath diameter, near-infrared spectroscopy, tympanic membrane displacement, visual-evoked potentials, some other measurements of the optic nerve,

*Example of multimodal brain monitoring recording during plateau wave, extracted from Dias et al. [45].*

*Altered ICP pulse waveform indicating compromised intracranial compliance, extracted from Dias et al. [45].*

*DOI: http://dx.doi.org/10.5772/intechopen.94077*

the mentioned study [45].

of arterial blood pressure and heart rate (**Figure 5**) [45].

components (P1 < P2 > P3) showed altered ICC [45] (**Figure 6**).

#### *Intracranial Pressure Waveform: History, Fundamentals and Applications in Brain Injuries DOI: http://dx.doi.org/10.5772/intechopen.94077*

decreased volume-pressure compensatory reserve such as TBI and many other brain pathologies. It consists of sudden increases in ICP to peaks of 40-100 mmHg that persists for 5–20 minutes [14, 45, 46]. The study observed plateau waves in 44% of the patients and that abrupt increase of ICP above 40 mmHg was associated with an increase in amplitude of ICP pulse waveform and also with important decrease in CPP, cerebral blood flow and oxygenation, despite stable cardiovascular variables of arterial blood pressure and heart rate (**Figure 5**) [45].

When analyzing ICP pulse waveform during plateau waves, a statistically significant increase in amplitude and a change in its shape were noted. The ICP pulse components (P1 < P2 > P3) showed altered ICC [45] (**Figure 6**).

Although slight increases in ICP that last for short period are not usually associated with poor outcome, if plateau waves are sustained over 30 minutes it could have a negative impact on patient's recovery as intracranial hypertension compromises cerebral perfusion and implicates neuronal deterioration [14, 23, 42, 43, 46]. The analysis of ICP absolute values and waveform patterns over time could provide important information for early detection of ICH in TBI patients of the mentioned study [45].

The debate about how ICP waveform analysis could provide improved clinical benefit and a more actionable evidence to bedside addresses integrated metrics on brain's intrinsic compensatory capacity (autoregulation) and oxygenation, besides computational analysis of multiple continuous streams of neuro-monitoring data and equipment development to easily display this information [14, 23, 42–46].

In this way, non-invasive techniques are coming forward to give quick ICP information to neurocritical care team, including transcranial Doppler, optic nerve sheath diameter, near-infrared spectroscopy, tympanic membrane displacement, visual-evoked potentials, some other measurements of the optic nerve,

**Figure 5.** *Example of multimodal brain monitoring recording during plateau wave, extracted from Dias et al. [45].*

**Figure 6.**

*Altered ICP pulse waveform indicating compromised intracranial compliance, extracted from Dias et al. [45].*

*Advancement and New Understanding in Brain Injury*

**Classification Categories Examples** Clinical severity Mild GCS: 13–15

Pathoanatomic type Focal (one concise area) Skull fracture

Mechanism of injury Penetrating Gunshot wound/projectile

As a result of this intricate association the ICP may rise if intrinsic compensatory mechanisms are not preserved and the sustained hypertension can prevent adequate perfusion depriving the brain of oxygen and nutrition. The combination of all situations related to TBI mentioned above need specific and adequate management through the whole trauma assistance from the pre-hospital setting to the critical

Blunt Head rotation

Moderate GCS: 9–12 Severe GCS ≤ 8

Diffuse (widespread area) Diffuse axonal injury

Contusion Epidural hematomas Subdural hematomas Subarachnoid hemorrhage Intraparenchymal hemorrhage

Concussion

Jolt/blast

Chronic traumatic encephalopathy

Pierced object/weapons (knife, etc.)

Acceleration-deceleration

The main method of assessment and management of severe TBI is monitoring

Neurocritical care specialists routinely base their clinical reasoning looking at the absolute value of ICP – measured in mmHg or cmH2O – combined with imaging exams – CT-scan or MRI. However, the numbers may not translate the entire complexity of intracranial dynamic. It is suggested that the ICP waves and the study of its morphology could bring differential evidence of altered intracranial compliance and

A qualitative analysis of the ICP waveform [44] described the relationship between amplitude of ICP pulse wave, values of ICP, values of cerebral perfusion pressure (CPP), and the outcome of severe head-injured patients. Intracranial hypertension was evidenced by absolute values of ICP and CT-scan parameters. In those with fatal outcomes there was an increase in the ICP waveform amplitude along with an increase of ICP value up to 25 mmHg, however, above this value the amplitude began to decrease. This breakpoint trend in the amplitude-value relationship was not present in patients with good/moderate outcome. Thus, it is suggested that the physics involving ICP, CPP and parenchyma dynamics inside intracranial cavities was somehow translated into the waveforms, and its analysis and correla-

Another study [45] involving TBI patients described the ICP plateau waves characteristics using multimodal brain monitoring as well as calculated indices of brain compensatory reserve and cerebrovascular reactivity. Plateau waves are associated with working cerebrovascular reactivity and occur in situations of

and treatment of ICP. It is a level II-B of evidence recommended to reduce in-

care unit (CCU) and subsequent rehabilitation [36, 40].

tions could be a useful additional tool for outcome prediction.

hospital and 2-week post-injury mortality [23, 41].

changes of pressure regimen [14, 23, 42, 43].

**8**

**Table 1.**

*Different classifications of TBI.*

retina, and pupil, besides the routinely used imaging exams of CT-scan and MRI [23, 42, 43, 45]. There is also a new non-invasive method of ICP monitoring that provides morphological data of ICP waves and intracranial compliance, adding celerity to this multimodal scenario [47, 48].

It is well established that ICH is an important issue after TBI because of its relationship to overall outcomes and all guidelines recommend a comprehensive ICP assessment – either invasively or non-invasively. Information about absolute values and waveform characteristics of ICP may together contribute to direct optimal management of TBI and good patient care [23, 42, 43, 45, 47, 48].

### **4.4 Increased ICP outside ICU environment**

The brain constitutes approximately 80% of intracranial volume, and blood and CSF each account for 10% [49–51]. The first compensatory mechanism for maintenance of normal ICP involves displacement and reduction of the CSF compartment, reduction of CBF, and lastly, displacement of cerebral parenchyma causing herniation. The slower the increment in ICP, the more useful this regulatory system. Therefore, rapidly growing masses like malignant gliomas have a higher risk of causing brain herniation than slow-growing tumors like meningiomas or nerve sheath tumors [52].

Transient elevation in ICP, generally from 50 to 100 mmHg and 5 to 20 minutes, leads to plateau wave phenomena. It can occur spontaneously or start after coughing, sneezing, or changes in position. This transient intracranial hypertension period may be accompanied also by transient headache, transient alteration of the level of consciousness and focal deficits [49, 50].

Obesity and its relation with sleep apnea obstructive syndrome may show ICC impairment due to overnight hypercarbia leading to cerebral vasodilation. Also, this population is likely to develop chronic idiopathic intracranial hypertension. Moreover, hydrocephalus patients of any etiology, migraineurs, progressive neurological focal and/or gait disorders, all these situations mentioned here for outpatients practice raise the yellow sign on the need for ICC evaluation.


**1. Introduction**

Kanamycin is widely used in the treatment of animal infections, added as growth promoters or feed additives for preventive therapy [1]. The antibacterial mechanism of kanamycin is that it can irreversibly bind to the bacterial ribosomal 30S subunit and inhibit its protein synthesis [2]. Because of its potential ototoxicity and nephrotoxicity [3–6], the indiscriminate use of kanamycin will enhance bacterial resistance and cause kanamycin-residue accumulation in animal-derived food, which threatens human health. Therefore, the European Union has promulgated regulations on the maximum residue limits (MRLs) of kanamycin in different food matrices (100 μg/kg for muscle, 100 μg/kg for egg, 600 μg/kg for liver, 2500 μg/kg for kidney, 150 μg/kg for milk and 100 μg/kg for chicken meat) [7].

Kanamycin was isolated in 1957 [8]. It is a mixture of several closely related compounds, such as main constituent kanamycin A (>95%), as well as minor constituents kanamycin B, C, and D (<5%). The major components are shown in Figure 1 [9]. In addition, degradation products such as 2-deoxystreptamine and paromamine can also be present [10]. Kanamycin A and C are isomers, whereas kanamycin B and D have different functional groups [9].

**Figure 1.** Structure of kanamycin A, B, C, and D and amikacin.

#### **2. The Pre-Treatment Methods of Food Sample**

The key point of detecting kanamycin is to remove the impurities or extract kanamycin from matrices. The usual techniques for extraction and cleanup of kanamycin from matrices include protein precipitation, liquid-liquid extraction (LLE), and solid-phase extraction (SPE) [11]. Based on these techniques, pre-treatment methods for kanamycin detection in food samples are summarized as follows.


**3. Results**

### *3.1. Phenotypes of Swarna and DTY-IL under Reproductive Stage Drought Stress*

In the 2014 and 2015 dry season (DS) drought-stress field trials at IRRI, DTY-IL and Swarna, showed similar grain yield (Figure 1A) and plant height (Figure 1B) under irrigated conditions. Under reproductive-stage drought stress height was reduced in both genotypes to a similar degree (Figure 1B). While yield was reduced in both lines under drought, DTY-IL achieved about double the average grain yield compared to Swarna (Figure 1A). In addition, three weeks after draining the field, Swarna showed clear signs of leaf rolling. Leaf rolling was also observed for Swarna in the greenhouse experiment. While there was no visible di fference in flag-leaf morphology between both genotypes under the well-watered condition (Figure 1C), a prominent leaf rolling phenotype in Swarna was observed after ten days of drought stress with complete leaf-rolling on the 12th day of drought stress (Figure 1D).

**Figure 1.** The effect of reproductive stage drought on yield, height and flag-leaf morphology. The average grain yield ( **A**) and plant height (**B**) across the 2014 and 2015 dry season (DS) field trial of DTY-Il and Swarna under irrigated and drought condition ( *N* = 2–9). Flag-leaf phenotypes of the DTY-IL and Swarna under well-watered conditions ( **C**) and the leaf-rolling phenotype during drought-stress ( **D**) under controlled greenhouse conditions. The asterisks (\*) indicates a significant di fference (Student's *t* test, \* *p* < 0.01).

### *3.2. Generating a Transcriptional Map of the Moderate RDS Response in Rice*

Mapping rates ranged from 77.7–92.9% for flag-leaf and 87.9–92.8% for panicle (Table S1), covering 28,283 and 33,698 genes, respectively. Sample clustering and heatmap visualization of log2-transformed, normalized count data demonstrated clear separation between genotypes and treatments for both flag-leaf and panicle samples (Figures S2A,B and S3A,B). Principal components (PC) analysis showed that the first and second PC explained 93% of the total variation for flag-leaf (Figure S2C) and 81% for panicle tissue (Figure S3C). Biological replicates of each genotype-treatment combination clustered together and the treatment effect was greater than the genotype effect for both tissues (Figures S2C and S3C). An exception was a single Swarna panicle sample, which was removed from all further analyses as an outlier (Figure S4). The sum of the non-redundant/unique log2 normalized genes with significant changes of expression at FDR adjusted *p*-value ≤ 0.05 across four different contrasts were 17,616 and 18,614 for flag-leaf and panicle tissue, respectively. Pairwise DE analysis for all genotype-treatment combinations identified DEGs of significance for flag-leaf and panicle (Figure S5), and a Venn diagram was used to visualize three categories of unique and common responses in flag-leaf (Figure 2A and Figure S6A,B, Table 1) and panicle (Figure 2B and Figure S6C,D, Table 1).

**Figure 2.** Gene di fferential expression and identification. Venn diagram of di fferentially expressed

genes (DEGs) for flag-leaf (**A**) and panicle (**B**) tissues in Swarna and DTY-IL under reproductive drought stress (RDS) at a false discovery rate (FDR) adjusted *p*-value < 0.05 and −1 ≤ log2-ratio ≤ +1 (fold change ≥ 2 and ≤ 0.5). The three highlighted boxes for each tissue represent the common DEGs (red), unique to Swarna (yellow), and unique to DTY-IL (blue). SWAC = Swarna control, SWAD = Swarna under RDS, ILC = DTY-IL control, ILD = DTY-IL under RDS.

**Table 1.** A summary of GO enrichment analysis of differentially expressed genes (DEGs) common to both genotypes, and unique to DTY-IL and Swarna for flag-leaf and panicle tissues under RDS.

### 3.2.1. Expression Profiles of Drought-Responsive Genes in the Flag-Leaf Tissue under RDS

A total of 4180 genes were found to be drought-responsive in flag-leaves of both Swarna and DTY-IL (Figure 2A, Table 1). Gene ontology (GO) enrichment analysis showed functional enrichment among upregulated DEG for transcription, regulation of biological processes, and oxidation-reduction (Table S2-1), while the shared downregulated DEGs were mainly associated with transmembrane transport, localization, and post-translational protein modification (Table S2-2).

In Swarna flag-leaves a total of 515 (188 up- and 327 downregulated) genes were uniquely drought-responsive (Table 1). While no significant GO terms were detected for upregulated DEGs (Table 1), significant GO terms for uniquely downregulated DEGs were largely related to post-translational protein modification, photosynthesis, defense response, and programmed cell death (Figure S7A). Pathway enrichment suggested photosynthesis, ubiquinone, and other terpenoid-quinone biosynthesis, as well as glutathione-mediated detoxification II and tyrosine biosynthesis (Table S3), to be significantly downregulated. MapMan visualization supported the downregulation of cell wall, carbon metabolism, secondary metabolism, and light reaction in Swarna flag-leaves (Figure 3A and Figure S9A).

In DTY-IL flag-leaves 108 (74 up- and 34 downregulated) DEGs were uniquely drought-responsive. While no significant GO terms could be associated with downregulated DEGs, oxidation-reduction, response to stress, and response to stimulus were among the significant GO terms in upregulated DEGs (Figure S7B). Pathway enrichment suggested phenylpropanoid biosynthesis, dhurrin, xylan, and scopoletin biosynthesis, as well as detoxification of reactive carbonyls in chloroplasts to be uniquely upregulated under RDS (Table S3). This was supported by MapMan visualization, showing upregulation of cell wall, lipids, and secondary metabolism (Figure 3B and Figure S9B).

Numerous overrepresented *cis*-elements were found in the group of 327 promoters of uniquely downregulated DEGs in Swarna in flag-leaf, which are mostly involved as binding sites for dehydration responsive genes. These include ACGTATERD1, IBOX, PREATPRODH, MYCATERD1, MYCATRD22, CCAATBOX1, and MYB2AT (Table S4-1). The overrepresented motifs in a set of 74 promoters in the uniquely upregulated DEGs in IL in flag-leaf were mostly functioning upon induction of dehydration stress through the ACGTATERD1 motif (Table S4-2).

3.2.2. Expression Profiles of Drought-Responsive Genes in the Panicle Tissue under RDS

A total of 4799 genes were found to be drought-responsive in panicles of both Swarna and DTY-IL (Figure 2B; Table 1). Enriched GO categories of the upregulated DEGs related to post-translational protein modification and response to stress (Table S5-1), while enriched GO terms of the downregulated DEGs were mostly related to transmembrane transport, carbohydrate metabolic process, and localization (Table S5-2).

In Swarna panicles, a total of 487 (184 up and 303 downregulated) genes were found uniquely drought-responsive (Table 1). No significant GO terms were identified within the uniquely upregulated DEGs. For the uniquely downregulated genes, significant GO terms included oxidation-reduction as well as monooxygenase activity, tetrapyrrole, and heme-binding (Figure S8A). Significantly enriched pathways associated with downregulated DEGs were related to DNA replication, diterpenoid biosynthesis, phenylpropanoid biosynthesis, and photosynthesis (Table S6). MapMan visualization supported the downregulation of cell wall, lipids, secondary metabolism, amino acids, as well as carbohydrate metabolism (Figure 3C and Figure S9C).

In DTY-IL panicle 164 (108 up and 56 downregulated) DEGs were uniquely drought-responsive (Table 1). No significant GO enrichment was identified among the uniquely downregulated DEGs of DTY-IL. Prevalent GO terms of upregulated DEGs were related to protein amino acid phosphorylation as well as oxidation-reduction and carbohydrate metabolic processes (Figure S8B). The most significantly enriched pathways in the panicle tissue of DTY-IL upregulated DEGs were related to propanoate metabolism, methylerythritol phosphate pathway, diterpenoid biosynthesis, camalexin biosynthesis, and circadian rhythm in plants (Table S6). This was supported by MapMan visualization, showing upregulation of cell wall, lipids, secondary metabolism as well as amino acids (Figure 3D and Figure S9D).

Overrepresented *cis*-acting elements in the group of 303 promoters of the uniquely downregulated DEGs in Swarna in panicle tissue mostly involved in dehydration response like the ACGTATERD1, MYBCOREATCYB1, ABRELATEDRD1, and IBOX motifs (Table S7-1). Overrepresented *cis*-elements in a set of 108 promoters of uniquely upregulated DEGs in DTY-IL in panicle tissue were mostly related to dehydration response like the MYB2AT, MYBCOREATCYCB1, and ACGTABOX (Table S7-2).

### *3.3. Drought-Stress-Related Gene Modules within the Transcriptional Map*

WGCNA identified 21 distinct co-expressed modules with di fferent expression patterns in flag-leaf (designated as FL-M1 to FL-M21, capturing 17,616 genes) (Figure S10B,C), and 23 distinct modules for panicle network (designated as P-M1 to P-M23, capturing 18,614 genes) (Figure S10E,F).

More than 70% of genes were distributed in the FL-M1 and FL-M2, and P-M1 and P-M2 (common response between DTY-IL and Swarna under RDS and control) for flag-leaf and panicle networks, respectively (Table S8-1,S8-2; Figure S11), signifying a common response shared between Swarna and DTY-IL. Modules FL-M1 and FL-M2 in the flag-leaf network showed "localization" and "transport" as the most enriched GO terms (Table S9-1). In the panicle network, P-M1 genes were enriched for functions related to "RNA processing" while the P-M2 module was linked with "localization" and "transport" (Table S9-2).