*5.1. Sampling*

A total of 274 samples including 70 mango fruits, 40 mango juice, 8 mango pulp, 15 mango jam, 77 orange fruits, 35 orange juice, 10 orange pulp, and 19 orange jam were purchased in di fferent areas of Central Punjab (Pakistan) from January to December 2018. The mango (*Mangifera indica*) and orange (*Citrus sinensis*) fruits were collected in supermarkets and local markets from Faisalabad, and from cities north (Rawalpindi, Sargodha, and Sheikhupura) or south (Multan, Shorkot, Layyah, Toba Tek Singh, and Sahiwal) Faisalabad. The mango- and orange-based products (juice, pulp, and jam) were from commercial brands of nationwide distribution purchased from supermarkets, local markets, and general stores in Faisalabad district. All samples were stored in their original packages at 4 ºC until they were analyzed. The samples were opened and thoroughly homogenized.

#### *5.2. Chemicals and Reagents*

Acetonitrile (HPLC grade), glacial acetic acid, sodium chloride, and sodium carbonate were purchased from Merck. Ethyl acetate, methanol, sodium sulfate anhydrous (analytical grade), 5-hydroximethyl furfural (5-HMF), and patulin (5 mg of solid crystalline) were purchased from

Sigma-Aldrich (Saint-Louis, MO, USA). Stock solution of patulin 1 mg /mL was prepared in acetonitrile and stored at −4 ◦C. Necessary volumes of stock solution were taken for working solutions (5, 10, 30, 50, 70, 100 μg/L) in 0.1% acetic acid. PriboFast multifunctional cleanup columns MFC 228 (Pribolab Pte. Ltd., Singapore) were applied for the optimization of the purification process.

#### *5.3. Sample Preparation and Extraction*

The purchased fruit samples were chosen free from debris, washed with water, shade-dried, and cut into small pieces with sharp knife. A sample of about 500 g was homogenized using high-speed blender (Braun Blender Mix 2000, Marktheidenfel, Germany). Samples of products (juice, pulp, and jam) were taken directly from the original packages. Analytical method for PAT was based on AOAC method 995.10 with little modifications [31]. Homogenized samples (25 g or 25 mL in triplicate) were extracted twice with 50 mL ethyl acetate along with 2 g sodium chloride in 250 mL Erlenmeyer flask (Pyrex, Germany) by shaking at high speed in horizontal shaker (Gunther and Co, Bremen, Germany) for one hour. The upper organic layers were combined and cleaned up with sodium carbonate solution (1.5% Na2CO3). Cleaned extract was rapidly dehydrated with 10 g anhydrous sodium sulfate (Na2SO4) and filtered with Whatman No. 1 filter paper. For alternative second-time clean-up, 9 mL cleaned extract was passed through a multifunctional column MFC 228, and 4 mL purified extract was collected. The extracts were evaporated to dryness under a stream of nitrogen. The dry residue was immediately dissolved in 1000 μL of 0.1% acetic acid and passed through syringe filter (0.22 μm, Millipore, Darmstadt, Germany). The samples were analyzed for PAT by reverse-phase HPLC equipped with UV-Vis detector (SPD-10AS, Shimadzu, Japan) in isocratic mode. The injection volume was 20 μL, and total run time was 10 min for PAT analysis.

#### *5.4. Apparatus and HPLC Conditions*

HPLC comprised a delivery pump (LS-10AS), system controller (SCL-10A), column oven (CTO-10A), UV-Vis detector (SPD-10AS), and Communication Bus Module (CBM-101), Shimadzu, Japan. Separations were performed in a Discovery HS C18 silica-based column (250 × 4.6 mm, 5 μm particle size; Supelco, Bellefonte, PA, USA), maintained at 30 ◦C with a flow rate of 1.5 mL/min in isocratic mode. Mobile phase was a mixture of acetonitrile:water (10:90, *v*/*v*) filtrated with 0.45μm filter (Nylon 66 membranes filter, Supelco, Bellefonte, PA, USA). Cleaned sample extracts and standards were injected using a Rheodyne injector (20 μL loop) in reverse-phase system, and wavelength of detection was 276 nm. Chromatograms were received with CLASS LC-10 Acquisition software. Qualitative and quantitative determinations of PAT were made comparing the retention time and peak area of reference standards. For method validation, a test was performed to confirm the peak separation of PAT and its principal interference 5-HMF. A solution containing 2 μg/mL of 5-HMF and 2 μg/mL of PAT was prepared and injected into HPLC (Figure 3).

**Figure 3.** Chromatogram of 5-HMF (5-hydroximethyl furfural) and patulin standards.


**4.5 Application of model: Horse Hill**

The first topic to be assessed is the possibility that the resumption in February 2019 of production from the Portland reservoir in well HH1 caused the third 'burst' of Newdigate seismicity. This phase of production is inferred to have begun at 08:00 GMT on 11 February 2019 and caused earthquakes starting with the event (MW 2.5; MO 6.8 1012 N m) at 07:43 GMT on 14 February 2019 (located at TQ 22959 41543, after [1], at a probable depth of 2400 m), event H in **Figure 7**. The time delay in this instance was thus 3 days or 72 hours, for a separation of 3 km (**Figures 1, 7**), the HH1 well bottom being 2.85 km from nearest point where the top Portland sandstone is transected by the Newdigate Fault and 3.0 km from an updip projection of the hypocentre to this cutoff. This production took place at 220 barrels per day or 0.40 l s1 . The pressure drawdown during this phase has not been reported. However, as detailed in the online supplement, during the flow testing of the Portland reservoir in well HH1 in July–August 2018, the developer reported production at a sustained rate of 190 barrels per day or 0.35 l s1 (140–160 barrels per day being of oil), with stable bottom hole pressures 1.4 MPa below the initial reservoir pressure of 6.3 MPa; scaling in proportion gives a 1.6 MPa pressure drawdown at the HH1 well bottom in February 2019 (i.e., 1.4 MPa 220/190). Using Eq. (52), with ν = 0.25 and δσ = 10 MPa, gives L = 134 m; using Eq. (49) gives b = 18.9 mm.

earthquakes starting with the event (MW 2.8; MO 1.3 1013 N m) at 12:10 BST on 1 April 2018 (located at TQ 21992 41976, after [1], also at a probable depth of 2400 m), event B in **Figure 7**, with hypocentre close to the western or WNW limit of the seismogenic part of the Newdigate fault. The time delay in this instance was 9 days 4 hours or 220 hours. In a straight line, the BRX2Y well bottom is 7.2 km NNW of the point, updip from this hypocentre, on the top Portland cutoff of the Newdigate fault (between circa TQ 18440 48310 and circa TQ 22100 42200). However, as already noted, it is possible that the high throw on the Brockham Fault along this direct path blocks any high-permeability connection, in which case the actual path length between these end points, through localities farther west with low fault offsets (**Figure 8**), and around the western end of the Leigh Fault (**Figure 1**), might be >1 km longer. Using Eq. (52), with ν = 0.25 and δσ = 10 MPa, MO 1.3 1013 N m for the 1 April 2018 earthquake gives L = 166 m; using Eq. (49)

*Seismicity at Newdigate, Surrey, during 2018–2019: A Candidate Mechanism…*

*DOI: http://dx.doi.org/10.5772/intechopen.94923*

**Figure 14(a)** shows the predicted variation in fluid pressure in the 'beef' layer that is inferred to hydraulically connect the Brockham Portland oil reservoir and the Newdigate fault, calculated using Eq. (15). With the chosen set of parameter values, following 4 m3 of production the resulting pressure decrease at r = 8 km distance is

appropriate for 7-inch diameter casing), using Eq. (14) the pressure decline at the well bottom at the end of production would be 530 kPa if the production took 4 hours or 1.0 MPa if completed in 2 hours. Within an hour of this idealised 50 Pa step pressure variation affecting the Dinantian limestone, Δx would be 0.06 μm (**Figure 14(b)**). This would be sufficient to unclamp the Newdigate fault

pressure decrease at r = 8 km would peak, circa 9 days after this pulse of production, at 150 Pa (**Figure 14(d)**). Within three quarters of an hour of this idealised 150 Pa step pressure variation affecting the Dinantian limestone, Δx would be 0.14 μm (**Figure 14(e)**). This would be sufficient to unclamp the Newdigate fault by 150 kPa or 2.5% of δσ (**Figure 14(f)**). Evidently, for such a small change in the state of stress to have caused the observed seismicity, the patch of this fault that slipped on 1 April 2018 must have already been within a very small proportion of δσ

The final topic assessed will be pressure interference between the wells. Like the path from the Brockham well to the Newdigate fault, the most permeable connection, through 'beef', between the two wells will exceed the straight line distance; separation r = 10 km is adopted. Again calculated using Eq. (29), with DB = 10 m2 s

the 4 m3 of production from well BRX2Y would cause a maximum pressure decline in the vicinity of well HH1 of 50 Pa after a 1 month delay (**Figure 15(a)**). With

of 90 Pa after a 15 day delay (**Figure 15(b)**). Variations in HH1 bottom hole pressure of this order, developing and dissipating on timescales of weeks or months in response to attempts at production from well BRX2Y, would be extremely difficult, if not impossible, to recognise given the >1 MPa pressure variations caused by the production from this well. This is consistent with the statement by the OGA [5] that no pressure variation at HH1 was detectable in response to the pulses of

A second test is possible, given that 20 years of production at Brockham (followed by two years of shut-in, during 2016–2018) resulted in a bottom-hole

, this production would cause a maximum pressure decline

by 60 kPa or 1% of δσ (**Figure 14(c)**). With DB adjusted to 20 m2 s

1

, and kP = 200 mD, with rW = 88.9 mm (again

. Assuming hP = 3 m,

1 , the

1

predicted to be 50 Pa after 9 days for DB = 10 m2 s

of the Mohr-Coulomb condition for slip.

1

DB increased to 20 m2 s

production from BRX2Y.

**95**

1

**4.7 Application of model: interference between the wells**

gives b = 21.5 mm.

η = 0.9 mPa s, DP = 1.3 m2 s

**Figure 13(a)** shows the predicted pressure variation in the 'beef' layer that is inferred to hydraulically connect the Horse Hill Portland reservoir and the Newdigate fault. With the chosen parameter values, the resulting pressure decrease at 3 km distance is calculated using Eq. (8) as 6.5 kPa after 2.5 days and 8.7 kPa after 3 days. Taking hP = 11 m, η = 0.9 mPa s, D = 1.3 m2 s 1 , and kP = 35 mD (somewhat higher than the 20 mD reported by Xodus [26], with rW = 88.9 mm (appropriate for 7-inch diameter casing), using Eq. (14) the pressure decline at the well bottom, 2–4 weeks after the start of production, would be 1.6 MPa, as expected. If η were adjusted to 11 mPa s, to reflect oil rather than water, kP 430 mD would be required to match this pressure variation.

**Figure 13(b)** shows the variation in fluid pressure inside the Dinantian limestone alongside the seismogenic part of the Newdigate fault, as a result of the imposed pressure variation in the 'beef'. **Figure 13(c)** shows the corresponding variation in Δx, the poroelastic separation of the opposing faces of this fault, and **Figure 13(d)** shows the corresponding variation in ΔΦ. The upper limit to ΔΦ is taken as c δσ, or 6 MPa, the coseismic stress drop, δσ = 10 MPa, also being the increase in shear stress during a complete earthquake cycle; this value of ΔΦ corresponds (using Eq. (44)) to Δx = 0.005 mm. These conditions are taken as indicating the condition for an earthquake to occur on the specified patch of fault. It is evident that with the specified combination of parameter values, these changes will occur very rapidly, in just 15 minutes after the idealised step onset of the pressure variation in the fault. In reality, **Figure 13(a)** indicates a gradual onset of this pressure variation, rather than an abrupt step, so the calculated changes Δx and ΔΦ will occur gradually, leading to an earthquake at the calculated time, roughly three days after the start of production from the well. This modelling demonstrates that, with the parameter values adopted, production from the Portland reservoir by well HH1 has been readily able to cause seismicity on the Newdigate fault.

#### **4.6 Application of model: Brockham**

The second topic assessed will be the possibility that the 4 m3 of production from well BRX2Y on 23 March 2018, inferred to have started at 08:00 BST, caused

### *Seismicity at Newdigate, Surrey, during 2018–2019: A Candidate Mechanism… DOI: http://dx.doi.org/10.5772/intechopen.94923*

earthquakes starting with the event (MW 2.8; MO 1.3 1013 N m) at 12:10 BST on 1 April 2018 (located at TQ 21992 41976, after [1], also at a probable depth of 2400 m), event B in **Figure 7**, with hypocentre close to the western or WNW limit of the seismogenic part of the Newdigate fault. The time delay in this instance was 9 days 4 hours or 220 hours. In a straight line, the BRX2Y well bottom is 7.2 km NNW of the point, updip from this hypocentre, on the top Portland cutoff of the Newdigate fault (between circa TQ 18440 48310 and circa TQ 22100 42200). However, as already noted, it is possible that the high throw on the Brockham Fault along this direct path blocks any high-permeability connection, in which case the actual path length between these end points, through localities farther west with low fault offsets (**Figure 8**), and around the western end of the Leigh Fault (**Figure 1**), might be >1 km longer. Using Eq. (52), with ν = 0.25 and δσ = 10 MPa, MO 1.3 1013 N m for the 1 April 2018 earthquake gives L = 166 m; using Eq. (49) gives b = 21.5 mm.

**Figure 14(a)** shows the predicted variation in fluid pressure in the 'beef' layer that is inferred to hydraulically connect the Brockham Portland oil reservoir and the Newdigate fault, calculated using Eq. (15). With the chosen set of parameter values, following 4 m3 of production the resulting pressure decrease at r = 8 km distance is predicted to be 50 Pa after 9 days for DB = 10 m2 s 1 . Assuming hP = 3 m, η = 0.9 mPa s, DP = 1.3 m2 s 1 , and kP = 200 mD, with rW = 88.9 mm (again appropriate for 7-inch diameter casing), using Eq. (14) the pressure decline at the well bottom at the end of production would be 530 kPa if the production took 4 hours or 1.0 MPa if completed in 2 hours. Within an hour of this idealised 50 Pa step pressure variation affecting the Dinantian limestone, Δx would be 0.06 μm (**Figure 14(b)**). This would be sufficient to unclamp the Newdigate fault by 60 kPa or 1% of δσ (**Figure 14(c)**). With DB adjusted to 20 m2 s 1 , the pressure decrease at r = 8 km would peak, circa 9 days after this pulse of production, at 150 Pa (**Figure 14(d)**). Within three quarters of an hour of this idealised 150 Pa step pressure variation affecting the Dinantian limestone, Δx would be 0.14 μm (**Figure 14(e)**). This would be sufficient to unclamp the Newdigate fault by 150 kPa or 2.5% of δσ (**Figure 14(f)**). Evidently, for such a small change in the state of stress to have caused the observed seismicity, the patch of this fault that slipped on 1 April 2018 must have already been within a very small proportion of δσ of the Mohr-Coulomb condition for slip.


**Abstract**

The aberrant tyrosine phosphorylation, either due to constitutive tyrosine kinases (TKs) or to inactivation of protein tyrosine phosphatases (PTPs), is a widespread feature of many cancerous cells. The BCR-ABL fusion protein, which arises from the Philadelphia chromosome, is a molecular distinct and peculiar trait of some kind of leukemia, namely Chronic Myeloid and Acute Lymphoblastic Leukemia, and displays constitutive tyrosine kinase activity. In the chapter, we will highlight the milestones that had led to the identification of the BCR-ABL fusion gene and its role as the only molecular pathogenic event sufficient to elicit and sustain chronic myeloid leukemia. We will also discuss the effort made to unveil the molecular mechanisms of action of the chimeric tyrosine kinase that eventually lead to aberrant cell proliferation and impaired cell-death. Furthermore, we will also review the lesson learned from the selective inhibition of BCR-ABL which currently represent a breakthrough in the treatment of several tumors characterized by defective tyrosine kinase activity.

**Keywords:** chromosomal translocation, fusion gene, tyrosine kinase, leukemia, tyrosine kinase inhibitors, targeted therapy

### **1. Introduction**

The up-regulated enzymatic activity of tyrosine kinases (TKs) is one of the most frequent events in human cancers. Basically, it is attributable to three distinct molecular-genetic mechanisms covering either the overexpression, the activating mutations or eventually chromosomal translocations involving tyrosine kinase genes. Therefore, targeting the kinases harboring oncogenic properties has led to prominent changes in cancer clinical management. An outstanding contribute in achieving the goal has been offered by the BCR-ABL oncogene, whose story started more than half a century ago. In the 1960s a couple of scientists working in Philadelphia described a major chromosomal abnormality in patients affected by Chronic Myeloid Leukemia (CML) [1, 2]. The chromosomal aberration consisted of an acrocentric chromosome that was originally thought as the outcome of a chromosomal deletion. At that time, it was the first chromosomal abnormality unambiguously associated to a specific malignancy. With the improvement of the chromosomal banding techniques, it became clear that the chromosome

abnormality was a shortened chromosome 22. Among a chorus of skepticism and wonder at the beginning of the seventies that short chromosome, that it is now known as Philadelphia chromosome (Ph), was identified as the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22). However, we had to wait until the eighties to know that the exact molecular consequence of the t(9;22) was a fusion gene encoding for a chimeric protein displaying constitutively tyrosine kinase activity. Altogether these discoveries delivered an outstanding message whereby a disease was tightly linked to a single oncogene, BCR-ABL. Since then, dozens of translocations have been found in other cancers, including acute promyelocytic leukemia.

BCR-ABL is a peculiar protein for several of reasons: 1) it is a chimeric protein that is encoded by a fusion gene deriving from a reciprocal chromosomal translocation; 2) it is a constitutively active tyrosine kinase eliciting oncogenic signals, 3) it has been the first oncogene associated to a disease displaying dual properties either as driver and in sustaining the neoplasm evolution, and 4) it has been the first kinase to be selectively targeted with small molecules, thus paving the way for the development of a number of tyrosine kinase inhibitors (TKIs).

In the present chapter we are going to discuss the milestones of a story, started 60 years ago, which has happily led to the selective pharmacological inhibition of BCR-ABL. Hence, CML, whose diagnosis was before a death sentence, is now successfully cured in the vast majority of the cases.


*5.2. Biomass, Grain Yield, and Water Use E*ffi*ciency*

The correlation between grain yield, biomass, and water productivity values for two barley genotypes showed that the observed and simulated values are closely co-related, as evidenced by the high R2 values, i.e., 0.91, 0.93, and 0.89 for grain yield, biomass, and water productivity, respectively (Figure 2).

**Figure 2.** *Cont*.

**Figure 2.** Correlation between observed and simulated (**a**) biomass yield; (**b**) grain yield; and (**c**) water productivity compared with 1:1 line.

The correlation between observed and simulated values of biomass yield for two barley genotypes at three locations showed proximity (Figure 3), which indicates the excellent ability of the AquaCrop model to predict biomass yield under different agro-climatic conditions. The results also show that the sensitive barley variety at MED produces the lowest biomass for both irrigation water qualities. Similar trends were observed for grain yield, where the tolerant barley variety performed better than the sensitive variety regardless of the location and the quality of irrigation water.

**Figure 3.** *Cont*.

**Figure 3.** Simulated and observed biomass of (**a**) tolerant and (**b**) sensitive barley genotypes (dots represent observations; simulations are represented by lines).

## *5.3. Canopy Cover (CC)*

The maximum and minimum CC were 85% and 30% in the sub-humid and arid areas, respectively. The salinity induces a 10% reduction in the CC in the sub-humid environment and 5–30% in the dry climate of MED. CC reduction under saline irrigation water is less noticeable in the tolerant variety than the sensitive variety for both salinity levels. However, in the rainfed area of Beja, the growth of both varieties was comparable.

Figure 4 shows a strong correlation between measured and simulated CC values for both varieties of barley (R2 = 0.91 and R2 = 0.93). In general, a good match between the observed and the simulated CC was observed in all three locations. However, the model somewhat over-estimated CC in the rainfed environment of Beja and slightly under-estimated it in the other two situations.

**Figure 4.** *Cont*.

**Figure 4.** Simulated and observed canopy cover for (**a**) tolerant and (**b**) sensitive barley varieties.

#### *5.4. E*ff*ects of Soil Salinity*

The maximum soil salinity was in the arid and semi-arid areas irrigated with saline water, respectively. The soil salinisation dynamic depends on the salinity of irrigation water. However, in the rainfed area of Beja, we noted the absence of any salty issue.

Figure 5 shows that the simulated soil salinity trend in the root zone (up to a depth of 0.7 m) corresponds very well with the measured values under different saline water regimes across different environments throughout the growing season. The observed and modeled soil salinity correlated well, with an R2 of 0.96. Figure 5 shows that the model reliably simulated average root zone salinity when the crop is irrigated with low-salinity water (2 dS m−1). However, it slightly underestimated soil salinity under higher saline water conditions (13 dS m−1), particularly for the late growing season.

**Figure 5.** Simulated and observed soil salinity in the testing-cropping season under different saline water regimes and across different environments.