**1. Introduction**

Lubricant industry develops and produces lubricating oils and greases for the lubrication of machine elements such as rolling bearings. Lubricants are essential to reduce friction and thus energy consumption on the one hand, and to prevent premature failure of machine elements due to wear. Lubricants also play an important role in damping the vibrations in bearings and decreasing the dissipated energy by decreasing the friction coefficient in the contact. Most lubricants available on the market today are still produced petrochemically, i.e., on the basis of fossil raw materials. With considering the expected shortage of fossil raw materials and the associated increase in the cost of products based on fossil raw materials, the industry is increasingly focusing on products made from renewable raw materials and the necessary adapted manufacturing processes [1,2]. Furthermore, in Germany, approximately 50% of the mineral oil-based lubricants used are released, each year, to the environment as a result of leakage or accidents, which causes environmental pollution due to chemical materials [3]. The first step toward developing biodegradable lubricants is to design sustainable lubricants, which are made of bio-based materials as an alternative to chemical resources [1].

Bio-based lubricants, such as ester or castor oil, are already available on market, which can be applied as an alternative to mineral oils [4]. In a recent study a bio lubricant is applied in a journal bearing and the friction behavior is compared to a mineral and a synthetic oil. It was found that, the bio lubricant showed lower friction compared to the mineral lubricant, however the synthetic oil showed the lowest friction [5]. In particular, there has been a focus on vegetable oils, such as palm oil and sunflower oil., to find a

**Citation:** Vafaei, S.; Fischer, D.; Jopen, M.; Jacobs, G.; König, F.; Weberskirch, R. Investigation of Tribological Behavior of Lubricating Greases Composed of Different Bio-Based Polymer Thickeners. *Lubricants* **2021**, *9*, 80. https:// doi.org/10.3390/lubricants9080080

Received: 21 June 2021 Accepted: 13 August 2021 Published: 17 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

suitable bio-based lubricant. Vegetable oils in comparison with mineral oils are renewable, easy to obtain, environmentally friendly, economically cheaper and hence, sustainable [6]. There was also a suggestion for application of bio lubricants based on soybean oil improved by epoxy compounds, shown in [7].

Usually, a bearing grease is based on around 70 to 97% of base oil, up to 10% of additives and around 3 to 30 wt.% [8]. According to literature, it is accepted to call a material biogenic or bio-based if it is biodegradable or it is produced partly or completely from renewable raw materials, as defined in [9]. The thickener system can be either based on lithium soaps of fatty acids or petrochemical based oligomers or polymers such as polyureas. Although, bio-based lubricants can be an alternative to commercial petrochemical greases [10], they still need to be developed and a reliable application must be approved. Regarding data from Agency for Renewable Resources (Fachagentur Nachwachsende Rohstoffe e.V.-FNR) the available greases on market are maximum 85% bio-based, since the thickener system is normally not bio-based. To develop a full bio-based grease, base oil and thickener system must be made from renewable materials. Recently, there has been some works on the development of a complete bio-based greases. The results demonstrated in [11] reveal, that a cellulose pulp from the Eucalyptus Globulus plant was obtained as the raw material to produce a thickener system for a bio-based grease. The cellulose pulp was also optimized in another study as a suggestion for bio-based thickener production [12]. Moreover, in [13] it was shown that bio-based greases were developed based on castor oil and high-oleic sunflower oil with cellulose fibers as thickener. In other studies, friction coefficients of developed bio-based greases were comparable to the petrochemical greases [14–16]. Even the wear in bearing with application of bio-based greases were shown to be lower than petrochemical greases in [17]. Although, some biobased greases have been proposed recently, it becomes more and more difficult to consider economic limits and ecological goals at the concurrently Therefore, new suggestions for biobased greases are always needed [18]. An alternative for producing bio-based thickeners is to develop polymer-based thickeners, such as polyurea, from sustainable materials, due to their high abilities compared to raw materials. If the thickener system is a lithium soap of conventional high molecular weight fatty acids, it represents a bio-based thickener. The main differences between biogenically thickeners to non-biogenic thickeners is the origin of the raw materials.

Promising bio-based feedstocks for the production of lubricating greases include 1,5-pentamethylene diisocyanate (PDI) [19], 2,5-bisaminomethylenefuran (BAMF), 1,5- pentamethylene diamine (PDA) and 4,4-diaminodiphenylmethane (MDA). PDA can already be obtained from glucose as reported in [20] and can be reacted with phosgene to give PDI. In this way, PDI with a biocarbon content of up to 70% can be obtained. BAMF can also be synthesized on the basis of glucose [21]. Hydroxymethylfurfural (5-HMF) is first obtained as the base chemical and converted to BAMF in several steps. At the present time, MDA cannot yet be produced bio-based on a large scale. However, MDA is produced on the basis of aniline, the sustainable production of which is already being worked on [22]. Thus, a substitution by bio-MDA is foreseeable in the near future.

Therefore, in this study we present the synthesis of bio-based polyurea thickener systems as well as the production method and chemical characterization of bio-based greases. Moreover, the focus of this study is to elucidate the tribological performance of the produced bio-based greases in comparison to a petrochemical reference grease. To evaluate the tribological performance, the results of film thickness and friction measurements on a ball on disc tribometer are discussed.

#### **2. Lubricants and Methods**

In the following the method of development and production of the bio-based greases are explained. Then to characterize the produced bio-based greases, methods of qualifications regarding tribological characteristics of the bio-based greases are explained.

#### *2.1. Bio-Based Grease Development and Production*

To produce the polyurea thickener systems, an in-situ polymerization of the monomers in castor oil was carried out. The reaction follows the mechanism of a polyaddition reaction. Three polyureas were prepared based on pentamethylene diisiocyanate (PDI) that was reacted with one of the diamines (a–c) (Scheme 1). As reaction vessel an ULTRA TURRAX® Tube Drive with ST-20 mixing vessel was used. The monomers were heated to melt and mixed in the oil at 100 ◦C. The PDI was added to the reaction mixture as the last component under stirring. After the addition of all components was completed, stirring was continued for another 2 min. A gel-like structure was typically formed within 10 s. The reaction progress was controlled by FTIR spectroscopy (Bruker Tensor 27, Berlin, Germany) shown in Figure 1. If the conversion was incomplete after two minutes and isocyanate bands were still detectable with the C-N band at 2270 cm−1, more stearyl amine was added to the reaction mixture until the C-N band completely disappeared. All three thickeners have a weight share of 15% of the grease. After cooling, the lubricating greases were homogenized by means of a three-roll mill.

**Scheme 1.** Reaction scheme of polyurea grease production with all components.

For all three polyurea systems the same monomer ratio was chosen, i.e., diisocyanate:diamine:monoamine was 1:0.75:0.25. This results in a degree of polymerization of 9 for the chain length of the polymer thickeners. The theoretical chain length was calculated using the CAROTHERS equation [23].

**Figure 1.** IR-spectroscopy of all three bio-based greases. Bio-based grease 1: PDI-BAMF (orange), bio-based grease 2: PDI-PDA (red) and bio-based grease 3: PDI-MDA (blue).

The successful production of the desired polymeric thickener structures was verified by 1H-NMR spectroscopy (Bruker DPX-400, Berlin, Germany). First, the respective grease was extracted with ethyl acetate in a Soxhlet extractor for three hours. The solid residue was dried under reduced pressure and the thickeners were obtained as yellow powder. Since polyureas are nearly insoluble in organic solvents, the powder was dissolved in concentrated sulfuric acid (98%). To determine the chemical shift *δ*, a capillary with deuterium oxide phase-separated was used as reference in 1H-NMR spectroscopy.

Using the bio-based grease 1 (PDI-BAMF system) as an example, the analysis of the 1H-NMR spectrum is shown below. Using the 1H-NMR spectrum of the bio-based grease 1 thickener (Figure 2) the successful synthesis of the chemical structure can be confirmed. With 1H-NMR the real number of protons for a structure can be determined, if the amount for one signal is exactly known. As can be seen from Figure 2 with exception of the protons (5) between the urea group and the furan ring, all protons are visible in the spectrum. These protons are overlaid by the signal of the reference D2O. Since the FTIR spectra (Figure 1) showed that no C-N band is present anymore in the greases, a complete reaction with the termination reagent stearyl amine can be assumed. Thus, there are two alkyl end groups per polymer chain and the protons of the methyl end group (1) can be seen at 0.66 ppm. Since this signal is baseline separated it can be used as an internal reference for the integration of all signals of the spectrum and refers to six protons per polymer chain, due to the fact that each methyl end group has three protons. This makes it possible to determine the exact number of protons for the other signals in the spectrum. For this purpose, the integral of the methyl group protons (1) is set to six. The protons of the CH2 groups of the alkyl chains (4) in the immediate vicinity of the urea groups also give a baseline separated signal. Therefore, this integral allows an exact determination of the number of repeating units within the polymer chain. For the PDI-BAMF system, four protons must therefore be subtracted from the integral (4), since they belong to the end group. This results in 36 protons. Since there are four protons for (4) per repeat unit, this value is divided by four. This results in an average repeat unit of nine for the polymer chains of the PDI-BAMF thickener. The experimentally determined value of the mean repeating units were in good agreement with the previously calculated using the Carothers equation [23].

**Figure 2.** 1H-NMR spectrum of the PDI-BAMF thickener (bio-based grease 1).

Furthermore, the relative flow limit of the three lubricating grease systems was investigated by using an MCR 302 rheometer (Anton Paar, Ostfildern, Germany). Oscillation

measurements according to DIN 51810-2 at 25 ◦C were performed. A plate-plate system with a geometry diameter of 25 mm was used. Forty-one data points were recorded for a shear deformation between 0.01 and 100%. The measurements were performed at a constant angular frequency of 1.59 Hz and a constant gap size of 1.000 mm. The flow limit was determined by the intersection of the logarithmically plotted curves from the storage and loss modulus. It was shown that castor oil is a suitable candidate regarding stability of produced grease and tribological characteristics such as friction coefficient [13,14,24]. Thus, castor oil was chosen as the base oil to produce the bio-based greases.

#### *2.2. Qualification of Bio-Based Greases*

The thickener systems and the stable bio-based greases were synthesized at TU Dortmund. The produced bio-based greases and grease properties are listed in Table 1. All bio-based greases had NLGI-class 2 as the result of investigations on penetration depth given in Table A3 in Appendix A. Furthermore, a petrochemical grease is also used for comparison the bio-based greases, which is also listed in Table 1.

**Table 1.** Summary of the bio-based greases and tested lubricants. PAO = polyalphaolefine.

Tribological tests were performed at the Institute for Machine Elements and Systems Engineering (MSE) of RWTH Aachen University to characterize the greases. For this purpose, the film thickness in the EHD contact was measured on a steel ball-on-glass disc EHD2-Tribometer from PCS Instruments as shown in Figure 3. A polished steel ball with a diameter of 19.05 mm was pushed against a glass disc with 47 N. The resulting Hertzian pressure was aimed to be 700 MPa and the temperature during the measurements was set to 40 ◦C. The film thickness measurements were conducted based on the interferometry concept, which is described in [25]. This method of measurements was also used in the literature [26]. Film thickness measurements were performed under pure rolling conditions, where the sliding effects were not present in the contact.

**Figure 3.** EHD2-ball-disc tribometer of PCS Instruments.

As preparation for the film thickness measurements, the grease was distributed on the disc with a grease film height of approximately 0.1 mm using a grease distributer following the measurement procedure described in previous work by the authors [27]. Before the actual measurement, the grease was uniformly distributed on the rolling track in the contact area by applying a 15 min running-in phase at a constant rolling speed of 30 mm/s. During the film thickness measurement, the rolling speed was continuously increased from 4 mm/s to 1000 mm/s in approximately 45 min. For oils, the relation of film thickness over rolling speeds is known to be linear in double logarithmic diagrams [8]. Therefore, to compare the film thickness of greases over rolling speed to base oil, the film thickness was measured six times at 61 logarithmically distributed rolling speeds under pure rolling condition, i.e., slide-to-roll-ratio (SRR) of 0%.

As the next characterization method, developed bio-based greases were tested by mean of friction losses in the EHD contact. In order to do so, the friction measurements were conducted using the EHD2-ball-on-disc tribometer. In contrast to the film thickness measurements, the steel ball was pressed against a polished steel disc and a torque transducer measured the traction forces acting on the ball. The friction coefficients were calculated by dividing the traction forces by the normal forces given to the ball. The schematic representation of this procedure is shown in Figure 4.

**Figure 4.** Schematic of the steel ball on steel disc friction measurements on EHD2 tribometer.

The material and roughness properties of the glass disc and steel disc and steel balls for measurements are listed in the Table 2.

The lubricant conditions in the rolling contacts can be determined based on the definition of lambda ratio as in [29,30]:

$$
\lambda = h\_0 / \sqrt{\left(R\_{q,1}^2 + R\_{q,2}^2\right)} \tag{1}
$$

where *h*0 is the film thickness and *Rq* is root-mean-square roughness of contact bodies.

According to [29,30], for values of higher than 3 for lambda, fluid regime and separation of both contact bodies is expected. Knowing the roughness of glass disc and steel ball from Table 2, corresponding film thickness value of lambda 3 was calculated equal to 18 nm. Analog to steel all and glass disc, corresponding film thickness of steel ball and steel disc can be calculated as 23 nm. The values show very small film thickness corresponding to lambda = 3. Therefore, it was assumed that no mixed lubrication regime occurs during the film thickness and friction measurements for the whole rolling speed range.

A relative speed between ball and disc was set to induce friction in the contact for friction measurements. Therefore, an SRR of 15% was adjusted. The greases are evenly distributed on the steel disc. Then, a running in phase was set before the actual friction measurement in analogy to the film thickness measurements. The EHD contact pressure for film thickness measurements was also 700 MPa and 40 ◦C. For each grease, both positive and negative SRR values were measured. This means that both scenarios of faster ball rotation than the disc and vice versa were measured. For each of the positive and negative SRR values, three independent measurements have been performed. In each test the rolling speed was increased from 15 mm/s to 1000 mm/s. The results were averaged to represent the trend of friction coefficient dependent on rolling speed.

#### **3. Results and Discussion**

In this section, three subsections are defined to demonstrate the results of characterization of bio-based greases. First, chemical characterization of the greases after production in labor is included. In the second subsection, results of film thickness measurements are explained. The three bio-based greases and the reference grease were compared and evaluated regarding the film formation capability and the occurrence of starvation. In the third subsection, the friction coefficients of the different greases were evaluated dependent on rolling speeds.

## *3.1. Chemical Characterisation*

Through the knowledge of the exact chemical structure of the thickeners, it is possible to determine the proportion of bio-based carbons for all three thickener systems (Table 3). The castor oil used was completely bio-based and accounts for 85 weight percent of the total grease. The remaining 15 weight percent was thickener, as no additives were used. The PDI used has a bio-carbon content of 70%. The BAMF and PDA used were bio-based. Stearyl amine and MDA were used as petrochemical products. For the bio-based grease 1 thickener (PDI-BAMF), this resulted in a value of 64% bio-based carbon. Taking the weight percentages into account, the total value was 95% bio-based carbon. Similarly, for the bio-based grease 3 thickener (PDI-PDA), this resulted in a value of 62 percent bio-based carbon for the thickener and a total value of 94% bio-based carbon by weight for the system. For the bio-based grease 2 thickener (PDI-MDA), only 20% bio-based carbon was obtained for the thickener which results in 88% bio-based carbon by weight. In all three systems, the maximum proportion of bio-based carbon can thus be increased by at least 3%, and in the best case by as much as 10%, compared commercially available systems that are currently used.

**Table 3.** Overview of all analytical data of the three thickener systems.

A flow limit of 16% was determined for the bio-based grease 1. Bio-based grease 3 had a flow limit of 25% shear deformation, which was the highest value of the three systems. Bio-based grease 2 showed the lowest value with a flow limit of only 5% (Figure 5). The flow limit of the bio-based greases can be correlated with the chemical structure of their thickeners. Both bio-based grease 1 and 2 contain aromatic thickener systems. The presence of aromatics in the polymer backbone results in a reduction of the chain mobility and stiffening of the polymer chain that may also facilitate π-π-stacking between different chains [31]. The chain stiffening increases even further for the PDI-MDA system, hence the lowest yield point is expected here. The bio-based grease 3 does not contain aromatics in the polymer backbone of the thickener and thus has the highest chain mobility. This allowed the system to withstand shear stress from the geometry for longer, resulting in a higher flow limit. The correlation of chain mobility as a function of polymer backbone has already been observed in rheology for other applications [32] and is in agreement with the literature.

Additionally, the dropping points of all systems were determined (Table 3). For the bio-based grease 1 a dropping point of 226 ◦C could be determined. The bio-based grease 3 had a dropping point of 252 ◦C. For the bio-based grease 2 a dropping point of 193 ◦C was determined. Class 2 was determined for the NLGI class of all three systems.

**Figure 5.** Flow limit determination for bio-based grease 1 (purple), bio-based grease 2 (green) and bio-based grease 3 (blue).

#### *3.2. Film Thickness Measurements*

In the first set of tribological characterization we measured the film thickness on ball-on-disc tribometer by varying the rolling speed to identify the onset of starvation for each grease. The results of film thickness measurements are shown in Figure 6.

As a reference, film thickness measurement of castor base oil with a measured kinematic viscosity of 254 mm2/s at 40 ◦C is also shown. As stated before, three measurements were averaged to represent the trend of film thickness dependent on rolling speed. The aim of the development of bio-based greases was to identify the bio-based thickener and base oil combinations, which can be used in industry instead of potentially harmful petrochemical based greases and thus reduce harmful environmental influences. Therefore, a petrochemical reference grease was also used to carry out film thickness measurements in comparison to the bio-based greases.

As shown in the upper left diagram in Figure 6, up to the speed of 100 mm/s, film thickness of bio-based grease 1 was higher than base oil. This is caused by thickener effects, as already described in [33–36]. From a rolling speed of 100 mm/s, film thickness increases with further increasing rolling speed, similar to base oil behavior. This effect was the hydrodynamic film formation which mainly depends on the rolling speed and base oil viscosity. In the speed range between 100 mm/s to 1000 mm/s, the base oil dominates the film formation, and thickener effects, assumingly, play a minor role [37]. In slower speeds, up to 100 mm/s, it was shown that bio-based grease 1 had the same film thickness as the reference grease. From 100 mm/s, it was shown that the bio-based grease even higher film thickness than the reference grease, and with increasing the speed the film thickness difference also increases. The hydrodynamic behavior was not captured in reference grease. In upper right diagram in Figure 6, the film thickness of bio-based grease 2 was smaller than base oil curve up to 20 mm/s. The hydrodynamic behavior of bio-based grease 2 ranges from 20 mm/s to 150 mm/s. Above that speed, the film thickness decreases with increasing rolling speed. This may happen due to an insufficient amount of lubricant replenishment in contact [8]. As presented in lower left diagram in Figure 6, hydrodynamic behavior of bio-based grease 3 starts at 20 mm/s and no decrease below the base oil curve can be detected.

In the lower right diagram in Figure 6, three bio-based greases are compared. It can be seen that the bio-based grease 2 shows the smallest film thickness up to 70 mm/s among all bio-based greases. Probably, the effect of thickener in bio-based grease 2 was the least distinctive. Up to 70 mm/s, bio-based grease 1 shows around 100 to 300 nm higher film thickness compared to bio-based grease 3. Thus, bio-based greases 1 and 3 with thickener systems PDI-BAMF and PDI-PDA show a tendency to form higher separating films over

the tested range of rolling speeds in comparison to bio-based grease 2 with thickener system PDI-MDA.

**Figure 6.** Film thickness of bio-based greases over rolling speed compared to reference grease and base oil at hertzian contact pressure of 700 MPa at 40 ◦C with SRR = 0 on steel ball on glass disc tribometer: (**a**) film thickness of bio-based grease 1, reference grease and base oil over rolling speed; (**b**) film thickness of bio-based grease 2, reference grease and base oil over rolling speed; (**c**) film thickness of bio-based grease 3, reference grease and base oil over rolling speed; (**d**) film thickness of bio-based grease 1, 2 and 3 and base oil over rolling speed.

The results of the lubricant film thickness measurement also correlate with the chemical structure of the thickeners. As previously shown with the rheology measurements, the chain mobility also has a decisive influence on the formation of the lubricant film. The polymer chains of the PDI-MDA system (bio-based grease 2) have the highest chain stiffness, from which a small contribution of the thickener structure to the formation of the lubricating film can be deduced. The PDI-BAMF system (bio-based grease 1) also contains an aromatic structure, but has a higher chain mobility. The PDI-PDA system (bio-based grease 3) has the highest chain mobility and is, therefore, the most effective in film thickness formation.

## *3.3. Friction Measurements*

The second method of characterization of the bio-based greases in this paper was measuring the produced friction coefficients on EHD contact on steel ball on steel disc measured by EHD2 tribometer by varying the rolling speed. Friction measurements were also conducted on the petrochemical reference grease to have a comparison with the bio-based greases.

The results of friction measurements are shown in Figure 7. In the upper left diagrams of Figure 7, it can be seen that friction coefficient in higher speeds, up to 800 mm/s, was same between bio-based grease 1 and the reference grease. The higher scatter in results was also observed in lower speed due to small grease particles passing through the contact, as was also captured in [38]. As shown in the upper right diagrams of Figure 7, the bio-based grease 2 shows similar friction coefficients to the reference grease up to 30 mm/s. In average speeds, from 30 mm/s to 400 mm/s, friction coefficient of the bio-based grease 1 was lower than the reference grease, and by higher speeds, from 30 mm/s, show both greases again the same friction coefficients. From the lower left diagrams of Figure 7, it can be seen that the friction coefficient of bio-based grease 3 is lower than the reference grease in all speed ranges up to 1000 mm/s. The friction coefficient of bio-based grease 3 decreases even further with increasing rolling speed.

**Figure 7.** Average friction coefficient of bio-based greases over rolling speed compared to reference grease at hertzian contact pressure of 700 MPa at 40 ◦C with SRR = 15% on steel ball on steel disc tribometer: (**a**) average friction coefficient of bio-based grease 1 and reference grease over rolling speed; (**b**) average friction coefficient of bio-based grease 2 and reference grease over rolling speed; (**c**) average friction coefficient of bio-based grease 3 and reference grease over rolling speed; (**d**) average friction coefficient of bio-based grease 1, 2 and 3 over rolling speed.

The bio-based greases are compared among each other in lower right diagram in Figure 7. It can be generalized that bio-based grease 3 results in the smallest friction coefficient. Up to 200 mm/s, bio-based grease 2 and 3 lead to similar values. The increase of friction at higher speeds for bio-based grease 3 can be linked directly to the decrease of film thickness and the occurring starvation at those speeds.


*2.2. Acetylation Changes Tau Phase Behavior*

LLPS has recently been observed for wt and hyperphosphorylated Tau, and truncation mutant (K18) [35–38]. At low salt conditions (5 mM sodium phosphate, pH 7.8), we observed near-instantaneous formation of wt Tau droplets (Figure 2A). Subsequent fusions indicate the liquid nature of the wt Tau droplets. We characterized the protein concentration (2.5–20 μM) and salt concentration (0–250 mM NaCl) dependencies of wt Tau LLPS. Consistent with the literature [37,38], higher salt concentrations disfavor LLPS and higher protein concentrations favor LLPS (Figure 2E). For the case of Ac-Tau, we observed a dramatic reduction in droplet formation (Figure 2B,F). Similar results were also observed when LLPS experiments were performed with wt Tau or Ac-Tau in the presence of a crowding agent (10% PEG 8K, 200 mM NaCl, 10 mM acetate, 10 mM glycine, 10 mM sodium phosphate, pH 7.5; Figure 2C,D). Thus, independent of the presence or absence of crowding, the hyperacetylation of Tau disfavors LLPS.

**Figure 2.** Acetylation disfavors Tau liquid-liquid phase separation (LLPS). Droplet formation of wt Tau versus hyperacetylated (Ac-Tau), respectively, in the absence (**A**,**B**) and presence of the crowding agent PEG-8K (**C**,**D**). Phase transition maps of Tau versus Ac-Tau (in the absence of crowders), respectively (**E**,**F**). The diagrams present protein and salt concentration dependencies of droplet formation. Blue open circles represent conditions of minimal droplet formation (<5 droplets/frame); small red solid circles represent 5–20 droplets, medium red solid circle 20–100 droplets, and large red circle >100 droplets. The bars in (**A**–**D**) represent 10 μm. See Section 3 for details.

Interestingly, even though both hyperphosphorylation and hyperacetylation decrease the overall pI of Tau, the two PTMs seem to have opposite effects on LLPS. In contrast to LLPS enhancement by hyperphosphorylation [36,37], hyperacetylation clearly disfavors Tau LLPS (Figure 2). Further experiments performed in identical or comparable conditions using the same Tau constructs, full-length or otherwise, are needed for a clear and direct comparison of LLPS behaviors of hyperphosphorylated, hyperacetylated, and wt Tau proteins. Nevertheless, we think that hyperacetylation disfavors full-length Tau LLPS by neutralizing the lysine positive charges, thereby affecting opposite-charge

attractions that help support Tau self- and mesoscale interactions. Our data also give direct support that electrostatics plays a major role in Tau LLPS.


**9. Treatment of special populations with direct acting antiviral regimens**

### **9.1. HIV/HCV-coinfected individuals**

Hepatitis C virus (HCV)-related liver disease is a major source of mortality in HIV-infected patients. Approximately one third of all patients with HIV are coinfected with HCV. Patients coinfected with HIV/HCV have shown lower rates of SVR with pegylated-interferon and weight-based ribavirin as well as more rapid progression of fibrosis than those with HCV monoinfection [103]. HIV/HCV-coinfected persons should be treated and retreated the same as persons without HIV infection, after recognizing and managing interactions with antire‐ troviral medications. Based on AASLD/IDSA/IAS-USA [93], the following precautions should be considered:

The management of HIV/HCV patients should take place in collaboration with an HIV practitioner. Special precautions should be taken when prescribing DAAs in patient on AIDS treatment to avoid under- or overdose in such patients as a result of drug-drug interactions. For example, ledipasvir increases tenofovir levels, concomitant use needs to be avoided in patients with CrCl below 60 mL/min. Because potentiation of this effect is expected when tenofovir is used with ritonavir-boosted HIV protease inhibitors, ledipas‐ vir should be avoided with this combination. Paritaprevir/ritonavir/ombitasvir plus dasabuvir should be used with antiretroviral drugs with which it does not have substan‐ tial interactions like atazanavir, enfuvirtide, lamivudine, emtricitabine, tenofovir, and raltegravir (and probably dolutegravir) [93].

The dose of ritonavir used for boosting of HIV protease inhibitors may need to be adjusted (or held) when administered with paritaprevir/ritonavir/ombitasvir plus dasabuvir, and then restored when HCV treatment is completed. The HIV protease inhibitor should be adminis‐ tered at the same time as the fixed-dose HCV combination. Simeprevir should only be used with antiretroviral drugs, with which it does not have clinically significant interactions like raltegravir (and probably dolutegravir), rilpivirine, maraviroc, enfuvirtide, tenofovir, emtri‐ citabine, lamivudine, and abacavir [93].

#### **9.2. Patients with decompensated cirrhosis**

In patients with Child-Pugh B or C cirrhosis awaiting transplantation, antiviral therapy may be offered on an individual basis in experienced centers, pending the presentation of more data in this population. It is possible that patients with decompensated cirrhosis who are not on a transplant list could benefit from an interferon-free treatment regimen. However, the safety and efficacy of an interferon-free regimen in patients with decompensated cirrhosis not on a transplant waiting list is unknown, and the impact on mortality in this group is not yet established. According to AASLD/IDSA/IAS-USA [93] and EASL recommendations on treatment of hepatitis C 2015 [91], the following medications can be used with high virological response >90%:

	- **•** Daily fixed-dose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) and ribavirin (initial dose of 600 mg, increased as tolerated) for 12 weeks is recommended for patients with decompensated cirrhosis.

**d.** Fixed-dose combination of paritaprevir (150 mg)/ritonavir (100 mg)/ombitasvir (25 mg) plus twice-daily dosed dasabuvir (250 mg) should not be used with efavirenz, rilpivirine,

**e.** Paritaprevir/ritonavir/ombitasvir with or without dasabuvir should not be used in HIV/

**f.** Simeprevir should not be used with efavirenz, etravirine, nevirapine, cobicistat, or any

The management of HIV/HCV patients should take place in collaboration with an HIV practitioner. Special precautions should be taken when prescribing DAAs in patient on AIDS treatment to avoid under- or overdose in such patients as a result of drug-drug interactions. For example, ledipasvir increases tenofovir levels, concomitant use needs to be avoided in patients with CrCl below 60 mL/min. Because potentiation of this effect is expected when tenofovir is used with ritonavir-boosted HIV protease inhibitors, ledipas‐ vir should be avoided with this combination. Paritaprevir/ritonavir/ombitasvir plus dasabuvir should be used with antiretroviral drugs with which it does not have substan‐ tial interactions like atazanavir, enfuvirtide, lamivudine, emtricitabine, tenofovir, and

The dose of ritonavir used for boosting of HIV protease inhibitors may need to be adjusted (or held) when administered with paritaprevir/ritonavir/ombitasvir plus dasabuvir, and then restored when HCV treatment is completed. The HIV protease inhibitor should be adminis‐ tered at the same time as the fixed-dose HCV combination. Simeprevir should only be used with antiretroviral drugs, with which it does not have clinically significant interactions like raltegravir (and probably dolutegravir), rilpivirine, maraviroc, enfuvirtide, tenofovir, emtri‐

In patients with Child-Pugh B or C cirrhosis awaiting transplantation, antiviral therapy may be offered on an individual basis in experienced centers, pending the presentation of more data in this population. It is possible that patients with decompensated cirrhosis who are not on a transplant list could benefit from an interferon-free treatment regimen. However, the safety and efficacy of an interferon-free regimen in patients with decompensated cirrhosis not on a transplant waiting list is unknown, and the impact on mortality in this group is not yet established. According to AASLD/IDSA/IAS-USA [93] and EASL recommendations on treatment of hepatitis C 2015 [91], the following medications can be used with high virological

**•** Daily fixed-dose combination of ledipasvir (90 mg)/sofosbuvir (400 mg) and ribavirin (initial dose of 600 mg, increased as tolerated) for 12 weeks is recommended for patients

HCV-coinfected individuals who are not taking antiretroviral therapy.

**g.** Ribavirin should not be used with didanosine, stavudine, or zidovudine.

darunavir, or ritonavir-boosted lopinavir.

raltegravir (and probably dolutegravir) [93].

citabine, lamivudine, and abacavir [93].

response >90%:

**9.2. Patients with decompensated cirrhosis**

**a.** Decompensated cirrhosis: genotypes 1 and 4

with decompensated cirrhosis.

HIV protease inhibitors.

94 Recent Advances in Liver Diseases and Surgery

**•** Daily sofosbuvir (400 mg) and weight-based ribavirin (1000 mg [<75 kg] to 1200 mg [>75 kg]) (doses need to be adjusted according to the patient's creatinine clearance rate and hemoglobin level) for up to 48 weeks is recommended for patients with HCV genotype 2 or 3 who have decompensated cirrhosis.

#### **9.3. Patients with HCV recurrence post liver transplantation**

Patients with posttransplant recurrence of HCV infection should be considered for therapy. Significant fibrosis or portal hypertension 1 year after transplantation could predict rapid disease progression and graft loss and could indicate the need for more urgent antiviral treatment. Interferon-free DAA can cure most liver transplant recipients with recurrent hepatitis C, including a majority of those with severe post-transplant liver disease. In addition to viral suppression, treatment also improves liver function. DAA treatment is generally safe and well tolerated, certainly more so than interferon-based therapy, although anemia remains a concern for people taking ribavirin. Drug-drug interactions may be important in the posttransplant setting. No clinically significant drug-drug interactions have been found between sofosbuvir, simeprevir, or daclatasvir on the one hand, and cyclosporine and tacrolimus on the other hand.

The following options proved to be useful in post-liver transplantation patients according to genotypes, with high virological response, waiting more data in near future [91,93,104,105]:

### **9.4. Patients with renal impairments**

For patients with creatinine clearance of >30 mL/min, no dosage adjustment is required when using simeprevir, sofosbuvir, daclatasvir, fixed-dose combination of ledipasvir (90 mg)/ sofosbuvir (400 mg), or fixed-dose combination of paritaprevir (150 mg)/ritonavir (100 mg)/ ombitasvir (25 mg) plus twice-daily dosed dasabuvir (250 mg) to treat patients with HCV infection. Simeprevir, daclatasvir, and the combination of paritaprevir, ritonavir, ombitasvir and dasabuvir are cleared by hepatic metabolism and can be used in patients with severe renal impairment [91].

EASL Recommendations on Treatment of Hepatitis C 2015 and AASLD/IDSA/IAS-USA 2014 guidelines on HCV treatment do not recommend sofosbuvir in patients with creatinine clearance of <30 mL/min or with ESRD until more data are available [91,93].

#### **9.5. Patients with acute HCV infection**

When the efficacy of the treatment of acute HCV infection was superior to the treatment of chronic infection, there was a strong impetus to identify and treat acute HCV infection with interferon [106]. The current availability of interferon-sparing HCV treatments that have high safety and efficacy reduces the advantage of early treatment of HCV infection. Until data documenting the efficacy and safety of treatment of acute hepatitis C with direct acting antiviral drugs are available, monitoring for spontaneous clearance for minimum of 6 months before initiating treatment is required. When a decision is made to treat patients after 6 months of acute infection, then the patient can be treated as described for chronic HCV [93].


**Declarations**

I, Mawande Sikibi, hereby declare that this paper is wholly my work and has not been submitted anywhere else for academic credit either by myself or another person. *Data Mining - Concepts and Applications*


**3. Conclusion**

102 Novel Aspects on Epilepsy

awareness. Recent ECG studies showed that every fifth epilepsy patients experiences abnormally slow heart rhythms during seizures, culminating in 16% with asystolie which is potentially lethal (Rugg-Gunn et al., 2004). For confirmed *KCNQ1* mutation carriers implantable pacemaker therapy should be considered in the context of abnormally slow heart rhythms, and considering the possible changes of brainstem parasympathetic outflow to the heart recently identified in knockin mice with Kv7.1 mutations (Goldman et al., 2009). As β-blockers are therapeutically recommended in LQTS1 in confirmed *KCNQ1* mutation carriers with the potential side effect of slow heart rates, it is important to carefully monitor

In addition, we have developed novel RyR2-specific compounds which permeate the bloodbrain barrier and inhibit both neuronal seizures and cardiac arrhythmias (Lehnart et al., 2008). Different from conventional ion channel blockers, RyR2 stabilizing compounds like S107 have no effect on physiological channel function and do not alter intracellular Ca2+ signaling (Lehnart, 2007). In addition, S107 has not shown any significant side effects against other ion channels or enzymes in large screening panels (Lehnart et al., 2008). Similar to LQTS1, β-blocker therapy is recommended in patients with CPVT1 and confirmed *RYR2* mutation carrier status due to the characteristic dependence of cardiac arrhythmias on sympathetic outflow to the heart. In summary, the neuro-cardiological implications of idiopathic epilepsies are potentially important in mutation carriers, and comprehensive phenotype and genotype risk profiling may lead to improved therapy and prevention of

**2.5 Summary of combined syndromes of neuronal and cardiac ion channelopathies**  Recent studies have for the first time identified previously unrecognized brain ion channelopathies as the neurobiological basis of generalized seizures. While the epilepsy phenotypes represent primary brain discharges, the same molecular defects precipitate lifethreatening cardiac arrhythmias. The following mechanisms have been proposed to underlie

Kv7.1 dysfunction in *KCNQ1* mutation carriers with LQTS1 may alter the propensity of neurons and cardiac myocytes to repolarize following depolarization by an action potential. This results in a decreased repolarization capacity in the brain and heart. In the brain, a decreased repolarization capacity during action potential firing may result in seizures and brainstem autonomic dysfunction of parasympathetic outflow leading to heart rhythm block and asystolie for example at the level of the cardiac AV node. Dysfunction of brain stem parasympathetic outflow to the cardiac sinus and atrioventricular nodes as a cause of asystolie in epilepsy expands previously described arrhythmia mechanisms, since fast ventricular arrhythmias are characteristically triggered by increased sympathetic outflow in *KCNQ1* mutation carriers. Because Kv7.1 mutations can cause dangerous cardiac arrhythmias, *KCNQ1* is a molecular candidate mechanism and risk factor of SUDEP. These findings establish *KCNQ1* in neuronal hyperexcitability and epileptogenesis. Hundreds of *KCNQ1* mutations have been identified mostly leading to loss-of-function phenotypes. However, the correlation between Kv7.1 channel defects and the clinical phenotype in different organs is variable, and not all mutations may cause epilepsy or only when a

*RYR2* mutations appear to cause increased neuronal burst activity of the hippocampal CA3 region and seizure activity as evidenced by synchronous bursting Ca2+ signals of principal

the heart rate behaviour in affected patients (Roden, 2008).

fatal arrhythmias in affected epilepsy patients.

epilepsy in combined neuro-cardiogenic syndromes:

permissive background or environmental condition exists.

Recent advances in ion channelopathy studies related to epilepsy have achieved important new insights about combined neuro-cardiogenic phenotypes, and mechanisms which may link dysrhythmia phenotypes of the brain and heart through the autonomic nervous system. Already, these insights have motivated further research about diagnostic strategies to improve risk prediction and prevention in patients with seizure disorders of unknown origin. In addition, novel therapeutic strategies which target the underlying molecular mechanisms are under development. Despite significant new mechanistic insight about Kv7.1 and RyR2 ion channel mutations as the cause of combined neuro-cardiogenic seizure and arrhythmia syndromes, many questions remain unanswered and require further study. These questions concern the specific brain regions and neuron types, which show a decreased threshold for aberrant network synchronization, such that prolonged depolarization or delayed repolarization may initiate primary brain seizures. In addition, arrhythmias in LQTS1 and CPVT1 are characteristically modulated by the autonomous nervous system. If the autonomous nervous system is also affected by the same gene mutations and exacerbates the cardiac and/or neuronal phenotypes needs further exploration through multidisciplinary research teams. Clearly, the new insights about neuro-cardiogenic dysrhythmias should motivate both clinicians and researchers to critically inquire complex phenotypes beyond the borders of a given research discipline.


*Edited by Blandina Bernal-Morales*

Emotional, physical and social well-being describe human health from birth. Good health goes hand in hand with the ability to handle stress for the future. However, biological factors such as diet, life experiences such as drug abuse, bullying, burnout and social factors such as family and community support at the school stage tend to mold health problems, affecting academic achievements. This book is a compilation of current scientific information about the challenges that students, families and teachers face regarding health and academic achievements. Contributions also relate to how physical activity, psychosocial support and other interventions can be made to understand resilience and vulnerability to school desertion. This book will be of interest to readers from broad professional fields, non-specialist readers, and those involved in education policy.

ISBN 978-1-78923-730-6

Health and Academic Achievement

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


**2.9 Cluster differentiation 146 Ag (CD-146)**

Cluster differentiation 146 Ag (CD-146) is a molecule of cell adhesion that belongs to the superfamily of immunoglobulins. It is identified as a progression marker for melanoma (melanoma adhesion molecule antigen) and breast cancer. The normal level of CD-146 in blood serum of healthy individuals is generally 309 μg L−1 [41].

For the identification of CD-146, a sandwich-based amperometric immunosensor was manufactured in which rGO-tetra ethylene pentaamine (TEPA) enhanced GCE antibody (Ab1) was immobilized as shown **Figure 10**. This improvement offered the electrode a large number of amino groups to improve the loading potential of antibodies. The secondary Ab was controlled with colloidal sphere TiO2 and nanoparticles Au/Pd and assay was conducted by calculating the amperometric reaction to electrocatalytic reduction of H2O2. However, the immunosensor displayed a wide linear range in 0.0050–20 ng mL−1 , a low limit detection value of 1.6 pg mL−1 [42].

Furthermore, biomimetic mussel-inspired polydopamine coating photoelectrochemical biosensing chip was constructed to detect CD146. The CdS/TiO2-ITO chip was designed using the electrodeposition process to deposit CdS on the TiO2-ITO chip. In addition, the PDA (polydopamine), developed by DA (dopamine) selfpolymerization, was anchored on the CdS/TiO2-ITO (cadmium sulphide/titanium dioxide-indium tinoxide) chip surface through its strong adhesivity and specific interactions such as electrostatic attractions or covalent bindings. Also, without using external crosslinkers, PDA/CdS/TiO2-ITO chips could be used for direct immobilization of antibodies. By measuring the photocurrent responses to different concentrations of CD146, quantitative determination of CD146 was achieved based on this principle. The photocurrent decreased linearly from 1 pg mL−1 to 20 ng mL−1 with an increase in CD146 concentration and a detection value of 0.3 pg mL−1 [43].

#### **Figure 10.**

*Schematic representation of the preparation of Au/Pd@TiO2-Ab2 (A) and immunosensor (B). Reprinted with permission from Ref. [43].*

### **2.10 Other important biomarkers**

Biomarkers such as tenascin-C (TN-C); p53 tumor suppressor protein (p53); DNA methyltransferase (DNA-MTase); estrogen receptor (ER); matrix metallopeptidase 9 (MMP-9); apurinic/apyrimidinic endonuclease 1 (APE1); mucin-like carcinoma-associated antigen (MCA); murine double minute 2 (MDM2); nuclear factor kappa B (NF-κB) are other important biomolecules that are known as tumor markers for breast cancer [44].

Tenascin-C is a large extracellular matrix protein newly expressed during tissue remodeling processes including angiogenesis, inflammation, and tumor growth. Tenascin-C is especially overexpressed in tumor tissues [45]. For the detection of TN-C a chemiluminescence (CL) based voltammetric immunosensor was prepared using carboxylated carbon nanoparticles (cCNPs) and conjugated with magnetic beads (MBs). In addition, an aptamer labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol was adsorbed to form labeled aptamer modified cCNPs-MBs on the surface of carboxymodified magnetic carbon nanoparticles. When the tenascin-C sample is applied, it will associate with the labeled aptamer to build a complex with the labeled aptamer. This complex is measured by CL whose amplitude is linearly linked to the tenascin C concentration by 1 pM to 1 nM and the detection value as 0.4 pM respectively [46].

Tumor suppressor protein (p53) is an important transcription factor in regulating cellular responses to stress factors. Loss of p53 activity induces tumor activation and gene mutation, resulting in a conformational alteration in the p53 protein structure [47]. The level of p53 protein in serum samples from cancer patients varies from 0.52 ± 0.23 ng mL−1 to 1.03 ± 0.59 ng mL−1 [48]. Another, nanocomposite based ultrasensitive electrochemical immunosensor for p53 was designed using polycysteine/graphene quantum dots/gold nanoparticle. In this study, p53-antibody was immobilized on a green and biocompatible nanocomposite comprising l-cysteine (P-Cys) as a conductive matrix and gold nanoparticles (GNPs) as a dual amplification component of graphene quantum dots (GQDs). This approach facilitated the linear analysis of p53 in the range from 0.0244 to 0.369 pM (SWV technique) and 0.195–50 pM (DPV technique) with a lower limit of 12.1 fM [49].

DNA methyltransferase (DNAMTase) is a widespread epigenetic alteration in both prokaryotes and eukaryotes and plays a crucial role in controlling gene expression, genomic stability and cell growth. Most tumors such as thyroid, liver, heart, prostate and breast cancer have over-expression of DNAMTase [50]. Further, DNAMTase was detected by label-free electrochemical biosensor for identification and inhibitor screening of methyltransferase activity based on graphene quantity and enzyme-catalyzed reaction. However, in this work, the modified HRP catalyzed the 3, 3′, 5, 5′-tetramethylbenzidine hydrogen peroxide-mediated oxidation resulting in an electrochemical signal production. The proposed biosensor achieved sensitivity detection of MTase activity within the range of 1–40 U mL−1 with a detection limit of 0.3 U mL−1 [51].

The biomarker of the estrogen receptor alpha (ERα) is a receptor and transcription factor with nuclear hormones. It controls gene expression and inhibits the proliferation and differentiation of cells in the target tissue. The occurrence of elevated ERα rates in the breast epithelium that implies an increased risk of breast cancer, indicating both the function of ERα in initiation and cancer progression [52]. For ERα detection, a electrochemical magneto immunosensing platform was developed taking an anti-human ERα antibodies modified SPCE. The antibody acquisition was immobilized on modified magnetic beads in carboxylic acid compounds, while the biotinylated antibody was labeled with a streptavidin-HRP conjugate. In addition, determining the target ERαprotein with a detection limit of 19 pg mL−1 evaluated the applicability of the integrated disposable magneto immunosensor [53].

**101**

*Current and Prospective of Breast Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.91151*

with a detection limit of 1 pg mL−1

controlled by an APE1 inhibitor and its isozyme discrimination [56].

tion limit is as low as 0.00518 U mL−1

**3. Future prospects and challenges**

100 ng mL−1

U mL−1

U mL−1

Matrix metalloproteinase 9 (MMP-9) is an extracellular 92 kDa protease belonging to a family of endopeptidases dependent on zinc and calcium. There was an improvement in expression of MMP-9 in a number of different tumors relative to healthy subjects, with an overall positive association between tumor aggressiveness and activity levels of MMP-9 [54]. For detection of MMP-9, SERS (surfaceenhanced Raman scattering) nano-tags integrated magnetic-separation biosensor was designed with whole blood. The silica coated Ag SERS nano-tags built as labels were used to identify MMP-9 in unprocessed blood samples in a quick and accurate MSB immune sensor. The results showed the sensitive and reproducible response constructed in whole blood to the concentration of MMP-9 in the range up to

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an intracellular multifunctional enzyme, also known as the redox impact factor 1. For detection of APE1, a novel electrochemical biosystem was designed by immobilizing antibody-APE1 modified on gold electrode. The technique skillfully incorporates immunoassay through an intricate template of enzyme activity study. The APE1 biosensor detec-

Mucin 1 (MUC1) protein is a membrane-associated glycoprotein containing 31 amino acids in the hydrophobic domain, 69 amino acids in the cytoplasmic domain and 20 identical amino acids per repeat in the extracellular domain. MUC1 is also a well-known tumor marker present in a range of malignant tumors [57]. For detection of MUC1, carboxylic group of disposable electrochemical immunosensors rich in graphene oxide for the identification of biomarkers with methylene blue using with human serum samples. The authors investigated highly conductive surfaces of carboxylic group rich graphene oxide on screen-printed carbon electrodes in this process. The established immunosensor demonstrated good detection range (0.1–2

), for MUC1 with outstanding linearity with a detection limit of 0.04

In recent years, the development of biosensors for biomarkers for breast cancer has received a lot of attention. However, the developments of biomarkers and the innovation of diagnostic tools for early detection of breast cancer are still in their early stages. While electrochemical immunoassays were very successful bio transducers, biomarkers established for breast cancer are needed to test their specificity, responsiveness and efficiency against the diagnostic standards created. The production and progression of these advanced cancer screening systems will aid in the early stages of accelerated clinical cancer diagnoses. Nonetheless, proposed detection approaches for biomarker detection of cancer necessarily require standardization of pre- and post-analytical protocols such as sample preparation, storage and optimization of experimental conditions for true validity of assays and more genuine output of the biosensor produced. Although very low LODs have been obtained by electrochemical biosensors, they typically convey multi-step mark strategies that complicate the experimental activity. For future works, the development another problem is that owing to its low accuracy and reliability, few portable electrochemical instruments are in clinical usage. Therefore, robust biosensor-based POC devices are required of ultrasensitive electrochemical label-free methods will be a great potential. Researchers must train the electrochemical biosensor to solve their reliability problems with a significant number of clinical samples. The development of wireless micro/nano electrochemical biosensors is an ideal option for in vivo

by using the differential pulse voltammetry (DPV) technique [58].

[55].

. The technique will monitor enzyme activity

#### *Current and Prospective of Breast Cancer Biomarkers DOI: http://dx.doi.org/10.5772/intechopen.91151*

*Molecular Biotechnology*

**2.10 Other important biomarkers**

markers for breast cancer [44].

from 0.52 ± 0.23 ng mL−1

detection limit of 0.3 U mL−1

Biomarkers such as tenascin-C (TN-C); p53 tumor suppressor protein (p53); DNA methyltransferase (DNA-MTase); estrogen receptor (ER); matrix metallopeptidase 9 (MMP-9); apurinic/apyrimidinic endonuclease 1 (APE1); mucin-like carcinoma-associated antigen (MCA); murine double minute 2 (MDM2); nuclear factor kappa B (NF-κB) are other important biomolecules that are known as tumor

Tenascin-C is a large extracellular matrix protein newly expressed during tissue remodeling processes including angiogenesis, inflammation, and tumor growth. Tenascin-C is especially overexpressed in tumor tissues [45]. For the detection of TN-C a chemiluminescence (CL) based voltammetric immunosensor was prepared using carboxylated carbon nanoparticles (cCNPs) and conjugated with magnetic beads (MBs). In addition, an aptamer labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol was adsorbed to form labeled aptamer modified cCNPs-MBs on the surface of carboxymodified magnetic carbon nanoparticles. When the tenascin-C sample is applied, it will associate with the labeled aptamer to build a complex with the labeled aptamer. This complex is measured by CL whose amplitude is linearly linked to the tenascin C concen-

tration by 1 pM to 1 nM and the detection value as 0.4 pM respectively [46].

to 1.03 ± 0.59 ng mL−1

0.195–50 pM (DPV technique) with a lower limit of 12.1 fM [49].

[51].

determining the target ERαprotein with a detection limit of 19 pg mL−1

the applicability of the integrated disposable magneto immunosensor [53].

based ultrasensitive electrochemical immunosensor for p53 was designed using polycysteine/graphene quantum dots/gold nanoparticle. In this study, p53-antibody was immobilized on a green and biocompatible nanocomposite comprising l-cysteine (P-Cys) as a conductive matrix and gold nanoparticles (GNPs) as a dual amplification component of graphene quantum dots (GQDs). This approach facilitated the linear analysis of p53 in the range from 0.0244 to 0.369 pM (SWV technique) and

DNA methyltransferase (DNAMTase) is a widespread epigenetic alteration in both prokaryotes and eukaryotes and plays a crucial role in controlling gene expression, genomic stability and cell growth. Most tumors such as thyroid, liver, heart, prostate and breast cancer have over-expression of DNAMTase [50]. Further, DNAMTase was detected by label-free electrochemical biosensor for identification and inhibitor screening of methyltransferase activity based on graphene quantity and enzyme-catalyzed reaction. However, in this work, the modified HRP catalyzed the 3, 3′, 5, 5′-tetramethylbenzidine hydrogen peroxide-mediated oxidation resulting in an electrochemical signal production. The proposed biosensor achieved sensitivity detection of MTase activity within the range of 1–40 U mL−1

The biomarker of the estrogen receptor alpha (ERα) is a receptor and transcription factor with nuclear hormones. It controls gene expression and inhibits the proliferation and differentiation of cells in the target tissue. The occurrence of elevated ERα rates in the breast epithelium that implies an increased risk of breast cancer, indicating both the function of ERα in initiation and cancer progression [52]. For ERα detection, a electrochemical magneto immunosensing platform was developed taking an anti-human ERα antibodies modified SPCE. The antibody acquisition was immobilized on modified magnetic beads in carboxylic acid compounds, while the biotinylated antibody was labeled with a streptavidin-HRP conjugate. In addition,

Tumor suppressor protein (p53) is an important transcription factor in regulating cellular responses to stress factors. Loss of p53 activity induces tumor activation and gene mutation, resulting in a conformational alteration in the p53 protein structure [47]. The level of p53 protein in serum samples from cancer patients varies

[48]. Another, nanocomposite

with a

evaluated

**100**

Matrix metalloproteinase 9 (MMP-9) is an extracellular 92 kDa protease belonging to a family of endopeptidases dependent on zinc and calcium. There was an improvement in expression of MMP-9 in a number of different tumors relative to healthy subjects, with an overall positive association between tumor aggressiveness and activity levels of MMP-9 [54]. For detection of MMP-9, SERS (surfaceenhanced Raman scattering) nano-tags integrated magnetic-separation biosensor was designed with whole blood. The silica coated Ag SERS nano-tags built as labels were used to identify MMP-9 in unprocessed blood samples in a quick and accurate MSB immune sensor. The results showed the sensitive and reproducible response constructed in whole blood to the concentration of MMP-9 in the range up to 100 ng mL−1 with a detection limit of 1 pg mL−1 [55].

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an intracellular multifunctional enzyme, also known as the redox impact factor 1. For detection of APE1, a novel electrochemical biosystem was designed by immobilizing antibody-APE1 modified on gold electrode. The technique skillfully incorporates immunoassay through an intricate template of enzyme activity study. The APE1 biosensor detection limit is as low as 0.00518 U mL−1 . The technique will monitor enzyme activity controlled by an APE1 inhibitor and its isozyme discrimination [56].

Mucin 1 (MUC1) protein is a membrane-associated glycoprotein containing 31 amino acids in the hydrophobic domain, 69 amino acids in the cytoplasmic domain and 20 identical amino acids per repeat in the extracellular domain. MUC1 is also a well-known tumor marker present in a range of malignant tumors [57]. For detection of MUC1, carboxylic group of disposable electrochemical immunosensors rich in graphene oxide for the identification of biomarkers with methylene blue using with human serum samples. The authors investigated highly conductive surfaces of carboxylic group rich graphene oxide on screen-printed carbon electrodes in this process. The established immunosensor demonstrated good detection range (0.1–2 U mL−1 ), for MUC1 with outstanding linearity with a detection limit of 0.04 U mL−1 by using the differential pulse voltammetry (DPV) technique [58].


*Minimally Invasive Lateral Approach for Anterior Spinal Cord Decompression in Thoracic… DOI: http://dx.doi.org/10.5772/intechopen.97669*

### **Table 5.**

*Literature review on mini-open lateral approach for symptomatic thoracic or upper lumbar disc disease: Spinal levels treated.*

### **Figure 8.**

*Preoperative sagittal (A) and axial (B) T2-weighted magnetic resonance images of a soft disc herniation at T10–11 in a 42 years old male with body mass index of 43.0. C: Postoperative sagittal x-rays showing decompression and placement of an expandable interbody cage in the same patient.*

*Minimally Invasive Lateral Approach for Anterior Spinal Cord Decompression in Thoracic… DOI: http://dx.doi.org/10.5772/intechopen.97669*

### **Table 6.**

*Literature review on mini-open lateral approach for symptomatic thoracic or upper lumbar disc disease: Complications.*

disc herniations and could be successfully repaired [7, 9]. Case no. 1 who had a calcified thoracic disc herniation had a small intraoperative cerebrospinal fluid leak repaired with onlay allograft and DuraSeal® (Covidien, Waltham, MA, USA). Other postoperative complications included 6 pleural effusions, two of which required interventional radiology placement of chest tube. Costovertebral neuralgia is usually treated with neuropathic pain medication such as gabapentin or pregabalin. Topical lidocaine can also be used.

### **3.4 Limitations**

The results of this study need to be interpreted with caution because of its retrospective nature and the limited number of cases reported. Although the outcomes were consistent with the literature, long-term follow-up would be necessary to better assess the risk of pseudoarthrosis and the persistence of resolution of symptoms.

### **4. Conclusion**

A larger number of case series have reported successful treatment of symptomatic TDH using the mini-open lateral technique. With a short length of stay for elective cases, a relatively low complication rate and improvement of motor function in the majority of patients, the mini-open lateral approach can be considered a safe and effective procedure for symptomatic TDH. Arthrodesis using expandable cages without additional anterior instrumentation can provide satisfactory shortterm outcomes. However supplemental anterior or posterior fixation should also be considered when significant pre-existing instability is suspected, when multiple contiguous levels are treated or when significant cage subsidence is noted during cage expansion.

*Minimally Invasive Spine Surgery - Advances and Innovations*


*2.3. Dictyophora indusiata Polysaccharide*

*Dictyophora indusiata* polysaccharide (DIP) is isolated from mushrooms with a molecular weight of 1132 kDa. The main linkage-type of DIP including (1→3)-linked α-L-Man, (1→2, 6)-linked α-d-Glc, (1→6)-linked β-d-Glc, (1→6)-linked β-d-Gal, and (1→6) linked β-d-Man. DIP could markedly facilitate the excretion of NO, TNF-α, and IL-6 from macrophages involved in complement receptor 3 (CR3) [22]. DIP has a latent treatment impact against colitis by increasing the level of *Bacteroidaceae* and *Enterobacteriaceae* in the colitis microenvironment. The administration of DSS-induced mice with DIP significantly reduced the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ) and enhanced antiinflammatory cytokines (IL-4, IL-10) [23]. The treatment of DIP with the DSS-induced mice restrains the biomarker of M1 macrophages CD86. It changed the macrophage subset distribution by reducing the production of M1 subsets from splenic macrophages and made deviate polarization to M2 subsets [24]. The macrophages have a tight connection to the inflammatory cytokines' generation. The M1 macrophages are connected to IL-6 as well as TNF-α, and M2 macrophages are associated with the generation of IL-10 [25]. DIP also can down-regulate the MPO level dramatically in a dose-dependent manner. As an inflammatory marker, the association between MPO and IBD is that it identifies the infiltration of inflammatory cells and tissue damage. Simultaneously, DIP also decreases NO activity and increases T-SOD levels. The latter would give rise to the production of NO [26], which triggers tissue damage and induces inflammatory responses during the occurring of IBD [18].

Recent studies have found that DIP not only decreased endotoxins, including lipopolysaccharides, but also maintained the normal level of tight junction (TJ) proteins expression including claudin-1, occludin, and zonula occludens (ZO)-1 in the antibiotic-induced mice [27]. Broad spectrum antibiotics have been demonstrated to cause gut dysbiosis [28] and destroy epithelial barrier integrity [29], thus raising the hazard of IBD occurrence in the neonates [30]. DIP could restore the function of the gut barrier. The gut mucosal barrier prevents the pathogens and toxins from invasion into the intestine, which has an essential function involved in maintaining the intestine in healthy condition. Additionally, DIP also inhibited the colonic tissue apoptosis in the ulcerative colitis mice by improving the expression of Bcl-2 and Bax [24].

#### *2.4. Flammuliana velutipes Polysaccharids*

Flammuliana velutipes polysaccharides (FVP) are comprised of glucans, galactan, xyloglucan, and xylomannan, whose backbone is α-(1→4)-d-glucose residues with branches consisting of (1→6)-linked α-d-glucopyranosyl [31]. FVP has various bioactivities including immunity regulation [32], anti-oxidant, anti-microbial, anti-tumor properties, and so on [33]. FVP could recover colon injury induced by DSS, and moderate the dysbiosis in the gut microbiota, such as by boosting the *Firmicutes/Bacteroidetes* ratio and enhancing the *Lachnospiraceae* abundance [34]. *Lachnospiraceae* is able to restrain intestinal immune homeostasis via promoting the SCFA production [35], which might explain the phenomenon that FVP increased the production of SCFAs in the gut.

#### *2.5. Pleurotus eryngii Polysaccharides*

Treatment of *Pleurotus eryngii* polysaccharides (PEP) could increase the abundance of *Porphyromonadaceae, Rikenellaceae, Bacteroidaceae,* and *Lactobacillaceae* on C57BL/6 mice. More importantly, PEP increased the SCFA production in the mice colon. SCFAs exert numerous functions in the mice including regulating gut immunity conditions, changing the mucus layer composition and restraining the homeostasis of the intestine [36]. At the same time, PEP inhibited the expression of ROS and NO in RAW264.7 macrophages, and sulphonated PEP showed more powerful anti-inflammatory activity [37].

A water-soluble polysaccharide (EPA-1) isolated from *Pleurotus eryngii* (99.7 kDa) consisting of mannose, glucose, and galactose with the ratio of 2.2:1.0:3.2 EPA-1 not only increased the manufacturing of NO and pro-inflammatory cytokines including TNF-α, IL-1, as well as IL-6, but also exhibited the improved expression level of phosphorylated p38, ERK, JNK, and NF-κB. It reflects the EPA-1 influence inflammatory response via the effect on MAPK and the NF-κB signal mediated pathway [38]. EPA-1 exerts intestine immunity to enhance its protective effect. The immunity cytokines including IL-6 motivated signaling pathways could promote the various leukocytes recruiting and activation [39], which is subsequently followed by possessing invading microorganisms and further diminishment of epithelial damage. Therefore, EPA-1 ameliorates IBD through moderate inflammatory cytokines. Above all these results have indicated that polysaccharides from *Pleurotus eryngii* have the function to alleviate IBD. Therefore, it is thought that PEP reduces the risk of IBD and benefits colitis recovery [40].

#### *2.6. Gracilaria Polysaccharides*

*Gracilaria caudata* polysaccharide consists of 1→3-linked-β-d-galactose and 1→4 linked-3,6-anhydro-α-L-galactose [41] with a molecular weight of 116 kDa [42]. Administrated *G. caudata* polysaccharide could significantly reduce neutrophil infiltration in acetic acid-induced mice via decreasing the expression of MPO levels together with restoring the colon from acetic acid injuries by reducing oxidative stress, including the diminishment of GSH consumption and iNOS expression in the gut [43].

Another species from genus of *Gracilaria* is *Graciliaria lemaneiformis*, the polysaccharides of *Graciliaria lemaneiformis* (SP), which also has sulfated groups. In the mice model developed by Ren et al. [18], SP was orally administrated 200, 400, 600 mg/kg/day for 3 weeks and the arresting weight loss following subsequently by the improved conditions of mice. Histological analysis showed that the colon structure, including the crypt number and muscular structure, was improved by raising the SP dosage, suggesting that a high dose of SP protects the goblet cells and crypt structure in the gut. SP also could repress the intestinal endotoxin and lipopolysaccharide-binding protein production, together with the reduction of TNF-α, IL-6, and IL-1β expression [44]. All of these markers indicate that SP alleviates the symptoms of IBD.

#### *2.7. Blidingia minima Polysaccharides*

*Blidingia minima* often grow in Subei Shoal in China and have similar morphology characteristics as *Enteromorpha* species. Polysaccharides extracted from *Enteromorpha* species have revealed various beneficial functions such as, for instance, anti-inflammatory and antioxidant activities [45]. Recently, Song et al. administrated *Blidingia minima* polysaccharides (BMP) to DSS-induced mice, and found a decrease in the expression of anti-inflammatory cytokine IL-10 and an increase in the production of pro-inflammatory cytokines including IL-1β and TNF-α. The expression of NF-κB, IκB-α, and AKT was also increased, thus, BMP might modulate the motivation of NF-κB and AKT signaling pathway to ameliorate IBD [46].


**3. Results**

In Figure 1, the CFE module developed with its morphological macroscopic appearance can be observed.

**Figure 1.** Picture of the esophageal flow control module – CFE; from left to right: upper and side views

The materials used in the manufacturing and packaging of the module that did withstand high temperatures, were sterilized with ethylene oxide. The materials (moulds) that withstand high temperatures were sterilized by steam autoclave.

### **3.1. Cadaver test**

176 Practical Applications in Biomedical Engineering

to determine location by means of image.

them, at the end of the experiment were evaluated.

*2.7.3. Video-endoscopy*

material.

**2.8. Exclusions**

**2.9. Statistical study**

confidence interval.

**3. Results**

be observed.

Radiographic follow-up of the animals was done of the cervical region on the lateral-right (in decubito) side, immediately after placement of the module, and every two days until its removal. The test was done in the radiology laboratory at the Veterinary School at UFG, according to the following protocol: 55 kV, 300 mA, 0.04 second of exposure in a right-lateral ducubitus position. A normal esophagus is not visible radiographically, because it is a close to collapsing organ and the module behaves like a foreign body (filled with gas), making it possible to visualize it radiographically. Thus, due to the radiopacity of the gas, it is possible

A video-endoscopy was performed on the 15th day, after withdrawal of the module. The procedure was analogous to that used during placement, utilizing the same endoscopic

There were no exclusions of the dogs, including those that expelled the modules. All of

The statistical hypothesis t-student test was used here to make a comparison between the groups (small samples). All discussions of the present study were carried out with a 99%

In Figure 1, the CFE module developed with its morphological macroscopic appearance can

**Figure 1.** Picture of the esophageal flow control module – CFE; from left to right: upper and side views

The materials used in the manufacturing and packaging of the module that did withstand high temperatures, were sterilized with ethylene oxide. The materials (moulds) that

withstand high temperatures were sterilized by steam autoclave.

*2.7.2. X-rays*

In this study, a guide needed for pulling the device through to the inside of the esophagus was developed. During the removal procedure, the external wall of the module was perforated for deflation and it was removed with endoscopic forceps.


**4. Conclusion**

Comparison of the geological and geophysical fields makes it possible to combine together different indicators of geodynamic processes. We have revealed the relationship between seismicity and deep structure, as well as the correspondence of seismicity to both the most geodynamically active structures and zones of concentrated tectonic stresses. According to the compiled unified catalog, the most geodynamically active area in the Barents Sea region is the Svalbard anticline, where the greatest concentration of tectonic stresses is observed on the MOR side and zones with higher values of heat flow are distinguished (70–80 mW/m2 on average).

The recorded single earthquakes in the basins and troughs reflect manifestations of recent tectonic activity in the region, probably as a result of commonly developed high-velocity inhomogeneities in the lower and upper crusts or accumulation and release of stresses in weakened zones. This conclusion is, however, quite conditional because of a small time interval for which seismic data on the Barents Sea region are available. Seismological monitoring is necessary in the future to verify or refute such conclusions.

Manifestations of single earthquakes should be marked as promising areas for searching and prospecting for hydrocarbon fields within the limits of the South and North Barents basins, St. Anna Trough, and western Barents Sea Basin. Our studies supplement existing data on the structure and tectonics of the Barents Sea region.


**3. Conclusions**

We presented a new microring resonator based on only one multimode waveguide. The critical control of the microring resonator can be achieved by using graphene-silicon waveguide. The proposed device has all characteristics of a traditional microring resonator. Some important parameters of the proposed device such as finesse, quality factor, etc. are also presented in this chapter. The device operation has been verified by using the FDTD. This microring resonator structure is very compact and can be useful for further applications in optical switching, filtering, and sensing.

### **Acknowledgement**

where Δ*λFWHM* is the resonance full width at half maximum and FSR is the free spectral range. The free spectral range is the distance between two peaks on a wavelength scale. By differentiating the equation *φ* ¼ *βLR*, we get

*Electromagnetic Propagation and Waveguides in Photonics and Microwave Engineering*

**Figure 9** shows the finesse and quality factor with different chemical potential at a radius of 5*μm*. We see that a maximum finesse and quality factor can be achieved

The normalized transmissions of the propose microring resonator in **Figure 1** at microring radii of 5*μm* and 50*μm* are shown in **Figure 10**. Here we assume that the chemical potential is *μc* ¼ 0*:*45*eV*. The simulations show that the exact characteris-

Finally, we use FDTD method to simulate the proposed microring resonator based on multimode waveguide. In our FDTD simulations, we take into account the wavelength dispersion of the silicon waveguide. A light pulse of 15 fs pulse width is launched from the input to investigate the transmission characteristics of the device. The grid sizes Δx = Δy = Δz = 20 nm are chosen in our simulations for

*Optical field propagation through the coupler for input signal presented at (a) port 1 and (b) port 2.*

*dλ* .

*ngL*, where the group index *ng* ¼ *neff* � *λ dneff*

*Transmissions of the microring resonator with two microring radii of* 5*μm and* 50*μm.*

tics of a single microring resonator can be achieved.

at a chemical potential of *μc* ¼ 0*:*57*eV*.

*FSR* ¼ *λ*2

**Figure 11.**

**58**

**Figure 10.**

This research is funded by the Ministry of Natural Resources and Environment of Vietnam under the project BĐKH.30/16-20.


**5.2 Industrial applications**

*Colloids - Types, Preparation and Applications*

rewet ability and vice-versa [1, 20].

using energy-related aspects together [37, 38].

**4.6 Inhibitor**

**4.7 Emulsification**

etc. [5].

**5. Applications**

**5.1 Biological activity**

hydrophobic group length in the chain present in the surfactant. Whereas, the fall in the concentration of gemini imidazoline surfactant resulted in a decreased in the

Surfactants are used as corrosion inhibitors; they act as a protector on metal surfaces from corrosion. The gemini surfactants are amphiphilic, hence develops an affinity at metal/metal oxide–water interfaces for adsorption phenomena which leads to creating a barrier on metals and metal oxides surfaces and inhibits corrosion [36]. Imidazoline plays a vital role in corrosion inhibition as the imidazoline ring bond with the planar alignment to the surface of the metal. Further studies showed that the imidazoline inhibits the corrosion by blocking, activating, and

A property of surfactants at the interface. Emulsification is a process that forms an emulsion between two immiscible liquids such as oil suspended in water [36]. Emulsification is an important property for gemini imidazolinium surfactants for the formation of the emulsion. The emulsification power of a surfactant varies with the length of the spacer group. It is directly proportional to the spacer chain length. Stable emulsions are required in drug formulation, cosmetics, solubilizations,

The antimicrobial activity of surfactants based upon the surface-active properties, hydrophobic chain length, and concentration. Gawali et al. (2019) synthesized the cationic Gemini surfactants and mentioned their application. The synthesized Gemini surfactants were assessed to check the biocidal activity contrary to a variety of bacteria such as Bacillus subtilis, *E. coli*, *Staphylococcus aureus*, and *P. aeruginosa*. The R = C7H15 [2-Octyl-1-diethylenediaminimidazoline based gemini surfactant (GSCTDH)] and R = C11H23 [2-dodecyl-1-diethylenediaminidazoline based gemini surfactant (GSLTDH)] compounds showed good microbial activity. The R = C13H27 [2-Tetradecyl-1-diethylenediaminimidazoline based gemini surfactant (GSMTDH)] surfactant was inactive for *Pseudomonas aeruginosa* but showed active behavior for Bacillus subtilis, *E. coli*, *Staphylococcus aureus*. The R = C15H31 [2-Hexadecyl-1-diethylenediaminimidazoline based gemini surfactant (GSPTDH)] compound showed biocidal activity against *E. coli*, *Staphylococcus aureus*. The GSCTDH surfactant compound containing the highest antimicrobial activity amongst the other Gemini synthesized compounds. As the compound showed the biocidal activity against the bacteria (gram-positive and negative), they can be considered to use as an antimicrobial agent in the form of surfactants. The test result showed the variation in the sensitivity of Gemini compound towards the gram-positive and gram-negative bacteria, they were more sensitive to gram-positive bacteria as compared to gram-negative bacteria. The reason behind that could be the behavior of the outer membrane of the bacteria which was less permeable in the case of gram-negative bacteria [39]. The surfactant having low critical micelle concentration values shows good corrosion inhibitor property due to their absorption quality

**74**

The gemini imidazoline surfactant with quaternary imidazolinium salts is used as a dispersant, emulsifiers, bleach agent, ant-static agent, and fabric softener as they show the better result as compared to the traditional surfactant and also have a mild effect on clothes, to eyes, and their biodegradability. Gemini imidazoline surfactant solubility is low in water [21]. They can be used for drug entrapment, oil recovery, also a probable vehicle for the transference of bioactive particles, for cleaning purpose, used as aerosol application [43].


**3.3 Relationship between the relative abundance of taxa and enzyme activities**

Axis 1 of the canonical correspondence analysis for bacterial community explained 80% of the variation (**Figure 3**). Two groups were segregated with regard

#### **Figure 3.**

*Canonical correspondence analysis (CCA) of bacterial phyla demonstrating the effect of wet/dry periods at the Guánica Dry Forest. Blue symbols represent wet periods and red symbols represent dry periods. Triangles, circles and plus sign represent tree species (Tabebuia heterophylla, Ficus citrifolia and Pisonia albida), respectively.*

#### **Figure 4.**

*Canonical correspondence analysis (CCA) of bacterial phyla and soil enzyme activities (Phosdi = phosphodiesterasae, ArylSul = aryl sulphatase, APho = alkaline phosphatase, AcidPho = acid phosphatase, Bcosi = β-glucosaminidase, and Bglu = β-glucosidase). Vectors represent enzyme activities.*

abundance due to tree species (**Table 2**). There were no significant differences in any of the diversity indexes with regard to tree species (**Table 3**). Sampling period exhibited an effect on bacterial species richness (P = 0.05) and on equitability (P = 0.0243). but not on the Shannon index. We found higher bacterial species richness during the wet period and higher equitability during the dry period

**3.2 Evaluation of indicator species analysis (ISA) for this forest ecosystem**

indicator species were found for the dry period (**Table 4**).

*Bacterial indicator species analysis (ISA) at the Guánica Dry Forest.*

The identification of species associated or indicative of groups of samples is a common aspect of ecological research [24]. Indicator species analysis (ISA) identified several bacterial (**Table 4**) species responsible for changes in soil microbial communities. Out of 185 bacterial OTUs, 10 served as indicator species for *Ficus citrifolia* during the wet period (**Table 4**). A total of 31 bacterial OTUs were identified as indicator species for *Pisonia albida* during the wet period. No bacterial

(**Table 3**).

**52**

**Table 4.**

*Microorganisms*

to dry and wet periods (**Figure 3**). Samples that correspond to the wet period were associated with acid phosphatase, alkaline phosphatase, β-glucosaminidase, β-glucosidase, and arylsulfatase (**Figure 4**), whereas samples corresponding to the dry period are associated with phosphodiesterase (**Figure 4**). Microbial taxa associated with wet samples were Bacteroidetes, Proteobacteria, and Tenericutes. Microbial taxa associated with dry sampling points were *Actinobacteria*, *Planctomycetes*, *Acidobacteria*, *Verrucomicrobia*, *Cyanobacteria*, and *Chloroflexi*.

under study have an accumulation of organic matter mainly composed of new and old leaf litter due to the harsh environmental conditions; this bacterial phylum is associated with the degradation of more complex substrates of SOM including lignin, which could explain their predominance in this forest soils [37–39]. Our data suggest that *Actinobacteria* were highly associated with phosphodiesterase at the Guánica forest. Other studies have also found that *Actinobacteria* populations (characterized by TRLFP) correlated with P content in semiarid environments [40]. Other studies have also demonstrated the production of phosphatases by cultivable soil *Actinobacteria* [41–43] serving as a further evidence of the trend observed in this study. I also found that Actinobacteria were the most abundant phyla during the dry period which is consistent with other reports. A similar trend was also found for a semiarid, high desert grassland north of Flagstaff, Arizona [44] and in another study, which evaluated African tropical forest soils and Chinese forest soils [45]. *Bacteroidetes*, the third most abundant phylum in this forest, are widely distributed in different habitats ranging from Antarctic ice, lakes, the gut of animals, and terrestrial environments [46]. Additionally, this phylum has the ability to withstand extreme desiccation conditions including droughts and UV light [47]. They have been found in fine dust traveling thousands of kilometers [48] and inside microaggregates; authors explain that in semiarid agroecosystems this could be a protection strategy employed by these microbes to endure extreme environmental conditions [48]. I found that *Bacteroidetes* were highly associated with the activities of two soil enzymes evaluated (acid phosphatase and alkaline phosphatase). These enzymes mineralize organic P forms into inorganic P forms. Another study provided evidence suggesting that environmental *Bacteroidetes* specialize in the mineralization of high molecular weight organic matter making them a key compartment for carbon fluxes and budgets in ecosystems [49]. *Bacteroidetes* are oligotrophic and are commonly associated with substrates rich in organic matter [50] as is the forest area where I based my study. The high abundance of *Bacteroidetes* could be associ-

*First Insights into the Resilience of the Soil Microbiome of a Tropical Dry Forest…*

*DOI: http://dx.doi.org/10.5772/intechopen.90395*

ated with the rich organic matter present in the sampling area.

**55**

**4.2 Influence of tree species on the bacterial populations in this forest**

Even though the most abundant bacterial phyla identified under all tree species were the same (*Proteobacteria*, *Actinobacteria*, and *Bacteroidetes*), some were specific for each tree species. Indicator species analysis (ISA) revealed that specific bacterial species were present under *Ficus citrifolia* and *Pisonia albida* only during the wet period. This trend suggests that these species assemblages may play an important role in the soil ecosystem processes under these specific tree species. One species was *Devosia* spp. whose OUT frequency was 14 (ind val = 0.62; p = 0.009) in soils collected under *Ficus citrifolia. Devosia* spp. forms part of the α-*Proteobacteria*; this genus is a non-rhizobia nodulating, nitrogen fixer [51]. Three different species of *Brevundimonas* spp. also occurred in high frequency under *Ficus citrifolia* during the wet period as indicated by ISA. This genus is actually known to produce phosphodiesterase, a group of enzymes involved in the degradation of organophosphorus [52]. During the wet period, 32 different OTUs were identified exclusively for *Pisonia albida*, and at least 7 of them had a frequency of 18. This high frequency suggests that indicator species may be playing an important role in the soil dynamics of *Pisonia albida*. For instance, three of the most frequent species are nitrogen fixers (*Microlunatus* sp., *Rhodospirillacease* spp*.*, and *Paenibacillus*), and *Pisonia albida* was the tree species with the highest total available nitrogen in this study (data not shown here). Even though we did not cultivate the indicator species nor have information regarding the physiology of the indicator species identified for


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