**2. GFP-based biomarkers**

The term biomarker has accumulated a variety of definitions over the years. Herein, bio‐ markers are defined as genetically encoded molecular indicators of state that are linked to specific genes.The utility of GFP as a biomarker was first demonstrated using GFP reporter constructs [45]. When GFP is used as a transcription reporter, a cellular promoter drives ex‐ pression of the fluorescent protein resulting in fluorescent signal that temporally and locally reflects expression from the promoter *in vivo*. In the initial experiments, GFP cDNA [46] was expressed from the T7 promoter in *E. coli* or from the mec-7 (beta tubulin) promoter in *C. elegans* [45].*E. coli* cells fluoresced and the expression in *C. elegans* mirrored the pattern known from antibody detection of the native protein.Subsequently, GFP transcriptional re‐ porters have been used in a wide variety of organisms; GFP expression has minimal effect on the cells and can be monitored noninvasively using techniques such as fluorescence mi‐ croscopy and fluorescence assisted cell sorting (reviewed in [47]).

GFP fusion proteins (generated by combining the fluorescent protein coding region with the coding region of the cellular protein) are used as markers for visualization of intracellular protein tracking and interactions (reviewed in [47-49]).The GFP moiety may be N-terminal, C-terminal or even internal to the cellular protein.The availability of a color palette of fluo‐ rescent proteins allows multicolor imaging of distinct fluorescent protein fusions in the cel‐ lular environment. GFP fusion proteins are a major component of the molecular toolkit in cell biology.

#### **2.1. Using GFP as an** *in vivo* **solubility marker**

GFP has been used as a genetically encoded reporter for folding of expressed proteins.Ex‐ pression of recombinant proteins in *E.coli* is a powerful tool for obtaining large quantities of purified protein; however, some overexpressed recombinant proteins improperly fold and aggregate. Manipulation of conditions to generate soluble protein can be a laborious proc‐ ess. Directed evolution can be employed to increase the solubility of the recombinant pro‐ teins, but detection of specific mutants with improved solubility is a challenge.However, GFP biomarking can be utilized to address this challenge.Since GFP chromophore formation requires proper protein folding and GFP folds poorly when fused to misfolded proteins, flu‐ orescence of a GFP fusion protein can serve as an internal signal of a specific soluble (not aggregated) protein [26]. When used as a folding reporter, GFP is fused C-terminally to the protein of interest using a short linker between the two protein domains.Detection of fluo‐ rescence indicates that GFP domain is properly folded and that the protein of interest there‐ fore must be soluble.If the protein of interest misfolds and aggregates, the fused slowfolding GFP aggregates along with it and fluorescence is not detected. Therefore, this folding reporter assay can be used as a screening tool for soluble recombinant proteins in the context of directed evolution.

Some LOO-GFPs also show interesting reactions to ambient light. LOO11-GFP (beta strand 11 omitted) does not bind strand 11 when kept completely in the dark, but does bind it upon irradiation with light [43]. Raman spectroscopy showed that, in the dark, the chromophore assumes a *trans* conformation, and that light induces a switch to the native *cis* conformation. After irradiation, the chromophore relaxes back to the *trans* conformation. Following up on this result, [44] showed that using a circularly permuted LOO10-GFP construct (beta strand 10 omitted) and introducing two synthetic forms of strand 10, the wild-type strand and a strand with a mutation to cause yellow-shifted fluorescence, light irradiation increased the frequency of "peptide exchange" between the two strand 10 forms. The presence of this pep‐ tide exchange suggests that the cis/trans isomerization of the chromophore requires partial

The term biomarker has accumulated a variety of definitions over the years. Herein, bio‐ markers are defined as genetically encoded molecular indicators of state that are linked to specific genes.The utility of GFP as a biomarker was first demonstrated using GFP reporter constructs [45]. When GFP is used as a transcription reporter, a cellular promoter drives ex‐ pression of the fluorescent protein resulting in fluorescent signal that temporally and locally reflects expression from the promoter *in vivo*. In the initial experiments, GFP cDNA [46] was expressed from the T7 promoter in *E. coli* or from the mec-7 (beta tubulin) promoter in *C. elegans* [45].*E. coli* cells fluoresced and the expression in *C. elegans* mirrored the pattern known from antibody detection of the native protein.Subsequently, GFP transcriptional re‐ porters have been used in a wide variety of organisms; GFP expression has minimal effect on the cells and can be monitored noninvasively using techniques such as fluorescence mi‐

GFP fusion proteins (generated by combining the fluorescent protein coding region with the coding region of the cellular protein) are used as markers for visualization of intracellular protein tracking and interactions (reviewed in [47-49]).The GFP moiety may be N-terminal, C-terminal or even internal to the cellular protein.The availability of a color palette of fluo‐ rescent proteins allows multicolor imaging of distinct fluorescent protein fusions in the cel‐ lular environment. GFP fusion proteins are a major component of the molecular toolkit in

GFP has been used as a genetically encoded reporter for folding of expressed proteins.Ex‐ pression of recombinant proteins in *E.coli* is a powerful tool for obtaining large quantities of purified protein; however, some overexpressed recombinant proteins improperly fold and aggregate. Manipulation of conditions to generate soluble protein can be a laborious proc‐ ess. Directed evolution can be employed to increase the solubility of the recombinant pro‐ teins, but detection of specific mutants with improved solubility is a challenge.However,

croscopy and fluorescence assisted cell sorting (reviewed in [47]).

**2.1. Using GFP as an** *in vivo* **solubility marker**

unfolding of the protein.

cell biology.

**2. GFP-based biomarkers**

12 State of the Art in Biosensors - General Aspects

Split GFP may also be used to assay folding and solubility of a protein of interest in vivo by "tagging" the recombinant protein with the smaller portion of the split GFP sequence, and expressing the larger portion separately or adding it exogenously. The small size of a pro‐ tein tag makes it less likely to interfere with the folding and function of the protein of inter‐ est.In the split GFP complementation assay a large fragment of GFP folding reporter (GFP1-10 ) is coexpressed with tagged GFP protein (GFP11-protein x) [50]. As shown in Fig‐ ure 6, neither GFP1-10 nor the GFP11-tagged protein fluoresce alone; however, if both com‐ ponents are soluble,GFP1-10 and the GFP11-tagged protein reconstitute the native structure and fluorescence.For successful implementation of the assay, directed evolution of super‐ folder GFP1-10 was required. This resulted in GFP1-10 OPT which has an 80% increased sol‐ ubility over the corresponding superfolder GFP1-10.GFP OPT contains 7 new mutations (N39I, T105K, E111V, I128T, K166T, I167V and S205T) in addition to the superfolder muta‐ tions [50].Directed evolution of GFP11 resulted in GFP11 optima tag that had the dual prop‐ erties of 1) complementation with GFP1-10 OPT and 2) minimized perturbation of the protein of interest. Note that full-length GFP OPT was subsequently found to be more toler‐ ant of circular permutation and truncation than superfolder GFP [40].

**Figure 6.** Mechanism of LOO11-GFP (GFP1-10) as an in vivo solubility indicator for proteins tagged with strand 11 (GFP11). Modified with permission from [50].

In addition to providing a less laborious method for detecting protein variants and reaction conditions for generating soluble recombinant protein, the split GFP complementation assay also serves as an assay of aggregation in living cells. For example, aggregates of the microtu‐ bule associated protein tau are found in neurofibrillary tangles but their role in the patholo‐ gy of Alzheimer disease and Parkinson disease is not clear [51].The split GFP complementation assay enables monitoring of the aggregation process in living mammalian cells [52,53] and was validated using GFP1-10 and GFP11-tau variants.Cells containing soluble tagged protein show visible fluorescence but aggregates have little or no fluorescence. Pro‐ tein aggregates of GFP11-tau sequestered the GFP11 tag, leading to decreased complementa‐ tion of GFP1-10 and decreased fluorescence. Thus the split GFP complementation assay using tagged-GFP tau showed that it could be used as an *in vivo* model for studying factors that influence aggregation.

#### **2.2. GFP biomarkers for single molecule imaging**

It is also possible to utilize GFP biomarkers for single-molecule localization, a form of superresolution microscopy. High affinity single chain camelid antibodies (nanobodies) to GFP can be used to deliver organic fluorophores to GFP tagged proteins that are in turn used in single molecule "nanoscopy." [54, 55]. This novel approach combines the molecular specific‐ ity of genetic tagging with the high photon yield of the organic dyes. Additionally, by vary‐ ing the buffer conditions used, many organic dyes can become photoswitchable. The small size of camelid antibodies and their high affinity allow for access to regions that are general‐ ly inaccessible to conventional antibodies and targets that are expressed at very low levels [56].

One should caution that the overexpression of FRET biomarkers in transgenic animals car‐ ries some concerns that this could lead to the perturbation of endogenous signaling path‐ ways and even retardation of animal development [57]. Additionally, in compact tissue, such as the brain tissue, cell type identification is particularly tedious due the diffused ex‐ pression of the biomarkers.


*2.6. Plasmid Construction and Plant Transformation*

The *Put2*-overexpression vector (full-length coding sequence of *Put2*) was constructed as previously described [30]. Gene loss-function of *put2* lines was generated through gene editing approaches. To generate the CRISPR/Cas9 vector, the two target sequences for *put2* were designed using the online software CRISPR-GE (http://skl.scau.edu.cn/ targetdesign/, accessed on 22 January 2022), which were inserted into two single guide RNA (sgRNA) expression cassettes through overlap PCR, followed by cloning into the pYLCRISPR/Cas9Pubi-H vector via Golden Gate ligation method [31]. The confirmed pFGC1008-*Put2*-3HA vector and pYLCRISPR/Cas9Pubi-H-*put2* binary vector were transformed into *Agrobacterium tumefaciens* strain GV3101 by electroporation after transgenic plants were generated with *Agrobacterium*-mediated cotyledon transformation of *Solanum lycopersicum* cv. Ailsa Craig via a method previously described [32]. Two separate homozygous T2 lines from mutation and overexpression lines were confirmed with Sanger sequencing and qRT-PCR. The *put2* mutants and *Put2*-OE plants were used in this study. The primers for vector construction are listed in Supplemental data S3 (Table S1).


**About the Editor**

#### **Jos ´e A. Orosa**

Jose A. Orosa currently works at the University of A Coru ´ na, E.T.S N ˜ autica y M ´ aquinas. He is ´ a Marine Engineering, Naval Architect and Mechanical Engineer and Full Professor at the University of A Coruna, Spain. His research projects are centred on energy efficiency and ship design and ˜ efficiency. He was identified as the second-most-relevant researcher in the last ten years (2006-2016) in Sustainability and Energy Efficiency by the study entitled "Worldwide Research on Energy Efficiency and Sustainability in Public Buildings (Sustainability 2017, 9, 1294; doi:10.3390/su9081294)".


*2.3.3 Rolling*

Unlike other alloys, rolling of titanium requires higher working pressure and extreme control in temperature. Cylindrical rollers are used to produce the strips, sheet and plate. In contrast, grooved rollers are employed in producing the rounds and other structural shapes. In sheet and plate rolling process, cross rolling is done to reduce the anisotropy in mechanical properties. Texture strengthening is less

**Table 1.** *Types of forging and its features [1].*

#### *Titanium Alloys - Novel Aspects of Their Manufacturing and Processing*

pronounced in the beta alloys compared to alpha alloys [1]. The lower rate of strain hardening of the beta alloy makes it more acquiescent to cold working.

In Ti-3.5Al-5Mo-6V-3Cr-2Sn-0.5Fe alloy, rolling and ageing in the sub-transus (alpha + beta field) temperature yielded a better combination of the strength and ductility compared to working in the beta field [13]. Sheet beta Ti alloys are amenable to cold rolling. Cold rolling has a strong effect upon mechanical properties. For example, Rosenberg [14] reported the effect of cold rolling on tensile strength, yield strength and ductility of Ti-15-3 alloy:

1.UTS (Rolled alloy) = UTS (un-rolled) + 0.75 × Percentage of reduction (%)

2.YS (Rolled alloy) = YS (un-rolled) + 0.65 × Percentage of reduction (%)

3.Ductility (Rolled alloy) = EL (un-rolled) − 0.65 × Percentage of reduction (%)

Two high roll mill and three high roll mill are commonly used for rolling titanium and its alloys.

#### **2.4 Thermomechanical processing**

Material processing performed with the aid of both mechanical force and thermal/ heat treatment can be termed as thermomechanical processing. The primary objective of this processing is to obtain a component in functional design with pre-determined microstructure and corresponding mechanical properties. Thermomechanical processing of beta Ti alloys can be done both above transus temperature (Super-transus processing) and below the transus temperature (Subtransus processing). Super-transus processing with hot deformation is optimised to obtain fine recrystallised beta grains. Sub-transus processing is optimised to obtain fine beta grains with controlled alpha phase morphology [12]. Size, volume fraction, morphology, and the spatial distribution of the alpha precipitates formed during the thermomechanical processing have a vital influence over the mechanical properties of the end product.

In Ti-15V-3Al-3Cr-3Sn alloy, Boyer et al. [15], showed the usefulness of thermomechanical treatment for attaining a wide range of tensile strength (from 1070 to 1610 MPa.)

#### **2.5 Heat treatment**

Heat treatment is the basic metallurgical process through which optimization of hardness, tensile strength, fatigue strength and fracture toughness can be achieved. All the metastable beta alloys are heat treatable to attain higher strength than alpha + beta alloys.

Duplex ageing treatment yielded a superior combination of mechanical properties with no precipitation free zone and finer alpha precipitation compared to single ageing in Ti-15V-3Al-3Cr-3Sn-3Zr [16] and Ti-3Al-8V-6Cr-4Mo-4Zr [17]. The rate of heating to ageing temperature was found to have a substantial effect on the evolution of microstructure and mechanical properties [18]. Choice of solution treatment temperature is important. For example, for Ti-1Al-8V-5Fe (Ti185), solution treatment near beta transus temperature leads to a highest tensile and yield strength [19].

Solution treatment followed by ageing in metastable beta alloys will lead to a microstructure consisting of soft alpha in the beta grain boundaries. Hence, this softer alpha phase may lead to the decline in the HCF behaviour [20] and tensile ductility by augmenting the intergranular fracture [17]. For example, Sauer and

**55**

*Processing of Beta Titanium Alloys for Aerospace and Biomedical Applications*

Luetjering [21] have also reported the adverse effect of alpha phase layers along the beta grain boundaries on the tensile and fatigue behaviour of Ti-5Al-2Sn-4Zr-

Modifying the surface is an effective and economical way to enhance the tribological and fatigue properties of the material. Thermo-chemical and mechanical

In order to enhance the surface hardness, wear resistance and near-surface strength, thermo-chemical surface processing techniques such as nitriding and carburising are employed. Among various thermo-chemical surface processing techniques, nitriding is extensively used. In this process, the nitrogen is fused into the titanium base alloy. Among the various technologies used for Nitriding, i.e., gas nitriding, laser nitriding, plasma nitriding, Ion nitriding and gas Nitriding are used widely [22]. Titanium nitrides will be formed on the surface as a result of the nitriding and these nitrides increase the surface hardness drastically and improve the tribological properties at the expense of the ductility of the material. Increased hardness due to TiN formation was made use in flap tracks of Military airplanes [23]. However, nitriding has a negative influence on the tensile strength and fatigue

Mechanical surface modifications such as shot peening, ball burnishing and laser peening are developed to enhance the fatigue behaviour of the target material by inducing the residual compressive stress and work hardening effect in near surface region. Both crack nucleation and crack propagation during fatigue loading were found to be affected by the surface modification treatment. However, surface roughness will be significantly increased at the end of the mechanical surface modification such as shot peening and this may lead to early crack initiation. Since 1970s, shot peening is being employed in enhancing the mechanical behaviour of Ti alloys in aerospace industries [24]. Schematic representation of shot peening is shown in the **Figure 2**. Shot peening of beta alloys, i.e. Ti-10V-2Fe-3Al and Ti-3Al-8V-6Cr-4Mo-4Zr yielded a marginal increase in the fatigue life compared to electro polished sample [25]. In LCB beta alloy, in order to compensate the residual compressive stress induced in the surface after peening, substantial tensile residual stress formed in the subsurface region and this deteriorated the fatigue behaviour compared to polished sample [26]. It is important to control the shot

Unlike shot peening and laser peening, roller burnishing reduces the surface roughness by stressing the surface with a roller ball with optimised pressure. Schematic representation of the roller burnishing is shown in the **Figure 3**. Roller burnishing of Ti-10V-2Fe-3Al beta alloy induced deeper and higher magnitude residual stress compared to shot peening. In roller burnishing of LCB beta alloy, higher the rolling pressure, deeper was the site for fatigue crack nucleation [27]. In Beta C (Ti-3Al-8V-6Cr-4Mo-4Zr) alloy, deep rolling ended up with deeper residual stress distribution compared to shot peening, but the magnitude of the residual stress remained high for the shot peened sample. A marginal increase in fatigue life

peening conditions to get the desired enhancement in fatigue life.

was achieved through deep rolling of Beta C alloy [28].

*DOI: http://dx.doi.org/10.5772/intechopen.81899*

*2.6.1 Thermo-chemical surface modification*

**2.6 Surface processing for aerospace application**

surface modification techniques are common in beta alloys.

4Mo-2Cr-1Fe (β CEZ).

strength of the material.

*2.6.2 Mechanical surface modification*

Luetjering [21] have also reported the adverse effect of alpha phase layers along the beta grain boundaries on the tensile and fatigue behaviour of Ti-5Al-2Sn-4Zr-4Mo-2Cr-1Fe (β CEZ).


**1. Introduction**

Bacterial infections are the second leading cause of death worldwide with an estimated 7.7 million deaths annually, of which 5 million are associated with antimicrobial resistance (AMR) to existing antibiotics [1].  Certainly, highly effective therapeutic interventions are needed more than ever to target bacterial infections and offset the rise in AMR. While various innovative approaches to discovering and developing new antibacterial agents have been proposed or undertaken such as the use of antibodies [2], aptamers [3], nucleic acid materials [4], nanoparticles of metal oxides [5–7], small synthetic chemical compounds [8,9], and others [10], antimicrobial host defense peptides (HDPs), which are part of the innate immune system, have been proven as a promising potential therapeutic strategy to confront bacterial infections and tackle the clinical threats of biofilm-forming drug-resistant strains due to the multiple advantages they offer [11]. HDPs are relatively small polymers (usually, 4–50 amino acid residues) that can be easily synthesized and modified at minimal costs. Unlike antibodies and small chemical compounds of nucleic acid-based drugs, antimicrobial HDPs often exhibit broad-spectrum activity against a wide range of bacteria species, including Gram-positive and Gram-negative pathogens, and usually, they tend to have a low propensity to develop antimicrobial resistance due to their multifaceted mechanism of actions [12,13]. These HDPs are also biodegradable, and tend to degrade easily; thus, they do not persist in the body and do not pose any undesirable side effects [13]. Furthermore, the ability of these antimicrobial peptides to disperse and eradicate mature bacterial

**Citation:** Etayash, H.; Yip, F.; Hancock, R.E.W. Impacts of PEGylation and Glycosylation on the Biological Properties of Host Defense Peptide IDR1018. *Pharmaceutics* **2023**, *15*, 1391. https://doi.org/10.3390/ pharmaceutics15051391

Academic Editors: Scavello Francesco, Amiche Mohamed and Jean-Eric Ghia

Received: 17 March 2023 Revised: 18 April 2023 Accepted: 27 April 2023 Published: 1 May 2023

**Copyright:** © 2023 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/).

biofilms and high-density bacterial infections both in vitro and in vivo gives them advantages over current antibiotics and other molecules as excellent antimicrobial and antibiofilm drug candidates [11]. Also, the ability of HDPs to act as anti-infective immune modulators with promising anti-inflammatory activity makes them clinically attractive candidates for multipurpose applications [14,15]. However, despite their advantages, as pharmaceutical products, HDPs endure several constraints that limit their ease of accessibility and use, including their systemic aggregation and toxic side effects typically at higher doses, in addition to their poor stability against blood-borne proteases [13,16]. While various approaches including, for instance, formulation strategies, sequence modifications and designing HDP mimetics have been explored to circumvent some of these shortcomings of HDPs, only limited successes for different peptides were achieved [13,17–23]. In this study, we decided to explore the special effects of both pegylation and glycosylation on the physiochemical properties as well as the biological activity properties of the HDP, IDR1018, which is a synthetic multifaceted peptide with immunomodulatory and antibiofilm activities [24,25]. Pegylation and glycosylation are modifications utilized in peptide/protein drug design and are often used to improve the stability and pharmacokinetic assets of potential drug candidates while reducing toxicities and potentially harmful side effects [26–29]. Pegylation is a process by which peptides are chemically conjugated to polyethylene glycol (PEG) in order to change their physicochemical or biological properties [30]. As PEG and its derivatives are inert, water-soluble, non-toxic and non-immunogenic, they are widely used to overcome limitations associated with biopharmaceutical products including water solubility issues, aggregations, toxicity against mammalian cells and immunogenicity [30]. Pegylation offers significant advantages for biopharmaceutical products, as attaching PEG improves proteolytic stability, helps mitigate the immunogenicity, increases resistance to bacterial-secreted enzymes, boosts blood circulation half-lives and enhances biodistribution as well as drug bioavailability [28,30]. Nevertheless, despite these advantages, pegylation is often associated with a partial or complete reduction in the antimicrobial activity of HDPs [30]. Glycosylation is a process by which a sugar moiety is chemically attached to biopharmaceutical molecules such as peptides, proteins, antibodies, etc., in order to change their physicochemical properties or produce better bioactive compounds [31]. As with pegylation, glycosylation can have a significant influence on the properties of HDPs, for example, modifying toxicities, resistance to proteolytic degradation, pharmacokinetics and dynamic properties [27,31,32]. However, as with pegylation, glycosylation does not always improve the antibacterial activity of HDPs since the attaching surges change the chemical structure, hydrophobicity and overall charge of the peptides, which can impact the insertion and interaction of peptides with bacterial membranes [31].

Indeed, the overall influence of pegylation and glycosylation on drug design is remarkable with many clinically accepted drugs being pegylated and glycosylated. Since little is known about the influence of both conjugations on the multifaceted properties of HDPs, especially the immunomodulatory functionality, we aimed in this study to look at their overall impacts by covalently modifying the antimicrobial HDP IDR1018 with short-chain PEG (PEG6) and a glucose moiety (N-acetyl glucosamine (GlcNAc)).

### **2. Materials and Methods**

#### *2.1. Peptide Synthesis, Pegylation, and Glycosylation*

IDR1018 peptide (sequence, VRLIVAVRIWRR-NH2) was purchased from GenScript (Piscataway, NJ, USA) at >95% purity. The pegylated IDR1018 [PEG6; VRLIVAVRIWRR-NH2] as well as the glycosylated IDR1018 [Glc-IDR1018; T(GlcNAc)VRLIVAVRIWRR-NH2], were obtained at ≥95% purity from Biomatik LLC (Wilmington, DE, USA) (Table 1).

**Table 1.** IDR1018 and its conjugated derivatives, HPLC, and mass spectra data.

HPLC, high-performance liquid chromatography; Ret. Time, retention time in minutes; MW, molecular weight; Calc., calculated; Obs., observed.

### *2.2. Aggregation Assay*

Solutions of IDR1018 peptide and the conjugates were tested for aggregation in saline (0.9% NaCl), 5% dextrose, and 10% RPMI tissue culture medium at 1 mg mL−1. The solutions were placed in 96 well plates and sterile water was used as a negative control. The turbidity was determined for each sample as described in earlier reports [19,33]. Similarly, the conjugates were screened for aggregation in the presence of sodium salts of polyatomic anions (citrate or phosphate) at various strengths (0.1−1000 mM). The % of aggregates was assessed relative to the OD600 of sterile water.

Furthermore, isolated peripheral blood mononuclear cells (PBMCs) seeded in 96-well flat-bottom tissue culture plates (Corning Inc., Corning, NY, USA) at a concentration of 2 × 106 in RMPI media were treated with the two conjugates at a final concentration of 32 μg mL−1, incubated for 4 h at 37 ◦C in 5% CO2, and visualized on a Nikon Eclipse TS100 microscope. Experiments were performed in triplicate in three independent experiments. Representative images of the microscopy are presented in Figure 1.


**11.2 Pembrolizumab**

Pembrolizumab prevents immune-cell deactivation and inhibition by sterically blocking the interaction of PD-1 and its ligands. Pembrolizumab was the first immune checkpoint inhibitor to be approved as a first-line treatment, as well as the first PD-1-targeted therapy. Pembrolizumab a dose of 10 mg/kg was administered every two weeks to patients with previously treated, advanced TNBC in the KEYNOTE-012 trial, which showed efficacy and an adequate safety profile [63]. The overall response rate was 18.5 percent of the 27 patients who were assessed for antitumor activity, with 17.9 weeks an average response time (**Table 3**) [63]. The KEYNOTE-086 trial is presently examining the use of pembrolizumab (200 mg per 3 weeks) in metastatic TNBC (NCT02447003). Cohorts A and B were presented in an oral session at the 2017 ASCO conference [64, 65]. Cohort A comprises of patients with TNBC who had advanced on at least one systemic treatment. Among the 170-patient cohort, 4.7% responded, and 7.6% accomplished disease control for 24 weeks or longer, which included stable disease, partial response, and complete response [66]. In addition, 0.6% showed an absolute response to pembrolizumab monotherapy, and 27% had a decrease in the target lesion size after the first dose. The KEYNOTE-086 trial's Cohort B included metastatic TNBC with PD-L1+ tumors, without having received some systematic treatment previously. 23% of the 52 patients in this cohort showed an objective responses [64]. The use of pembrolizumab as a primary therapy and the inclusion of PD-L1+ tumors as a criterion for inclusion may have contributed to the increased response in cohort B, with only 58 percent of the patients admitted had a cumulative PD-L1 positive composite score of >1 (**Table 3**) [64].


**Abstract**

Ways and possibilities for design, implementation and application of microcomputer-based embedded systems using open source hardware and software platforms are considered, proposed and described. It is proposed to use open source hardware and software microcomputer-based technologies for design and implementation of embedded systems in many practical needs and applications. Main advantages and possibilities of application and implementation of such embedded systems are considered and described. Two practically designed and implemented systems performing needed data acquisition and control are presented and described. Used technologies for realization of the systems and embedded applications of the solutions are described. Open source microcomputer boards, appropriate sensors, actuators and additional electronics are used for implementation of the systems hardware. Open source tools and programs and LINUX operating system are used for implementation of the systems software. Modular approach is applied in the systems design and realization. Easy system expandability, simplifying maintenance and adaptation of the system to user requirements and needs are enabled with such approach. Balance between functionality and cost of the systems was also achieved. Optimization according to user requirements and needs, low consumption of electrical energy and low cost of the system are main advantages of such systems compared with standard embedded systems. These systems are optimized and specialized systems for specific needs and requirements of users.

**Keywords:** microcomputer-based embedded systems, open source hardware and open source software, LINUX operating system, modular system design, low cost of system, low consumption of electrical energy, system specialization and optimization


*4.2. Summary*

KPR is a multi-agent multi-choice repeated game where players try to learn from their past successes or failures, utilizing publicly available information on the crowd sizes at different restaurant in the past to decide which restaurant to visit that day such that she would be alone there for being served the only prepared dish. Here, asymmetric case such that *λN* (*λ* < 1) agents are considered against *N* restaurants, for sufficiently large *N*. End of each day (iteration), we have measured social utilization for agents *f a*(*t*) = ∑*Ni*=1[*δ*(*ni*(*t*))/*λN*] where *ni*(*t*) denotes number of customers visiting *i*th restaurant on day *t*.

As shown in Figure 6 (for strategy I) and Figure 8 (for strategy II), the social wastage fraction (1 − *f a*) vanishes at the effective critical point *λc*(*N*) with the critical exponent *β* value near unity. In addition, from Figure 7 (for strategy I) and Figure 9 (for strategy II), we see that the the convergence or relaxation time *t*, required for *f a* to stabilize, divergence near the same critical points *λc*(*N*) for the respective strategies, with the exponent *γ* value about 1/2. Additionally, the finite size scaling analysis *λc*(*N*) ∼ *λc* + *const*.*N*−1/(*dν*), where *λc* corresponds to *λc*(*N*) for *N* going to infinity and *d* corresponds to the effective dimension, suggests the effective correlation length exponent *dν* value to be around 2 for both the strategies, as expected for such mean field (infinite range systems).

In Reference [81], we have studied the dynamics of the KPR game following the same two strategies for the case *λ* = 1. For *λ* = 1.0, where the critical points *λc* (for both the strategies) vanish, the universality class (values of the critical exponents *β* and *γ* were observed to be distinctly different, and this point needs further investigations. We may, however, note that, since at *λ* = 1, the number of both agents and restaurants are same (*N*), full social utilization (where *f a* = 1 = *f r*, occurring at *α* = 0+ for strategy I and at *p* = 1− for strategy II) induces an additional frustrating constraint in the collective choice dynamics involved here.

The KPR game models have been extended already and used to study real life problems, like resource allocation in Internet of Things [82], vehicle for hire [83], matching in mobility markets [84], etc. We hope the KPR game models will be utilized much more effectively in the context of much wider practical areas of collective learning dynamics and choices.

#### **5. Future of Econophysics: Some Perspective**

One often says that the main purpose of economic activity is to optimize the limited funds of labor and capital, natural and technical resources and capital resources, to satisfy our (practically) unlimited needs. "Economic science is therefore the science of efficiency, and as such, it is a quantitative science." [89] (also see Reference [90]). We have already argued [14] in Section 2 that epistemologically economics belongs to natural science (and not mathematics). It begins with observation which are to be analyzed using logic or mathematics and eventually should end in observation, as in all natural sciences. Since 1990s, most Universities of the world offer Science Graduation degrees (Bachelor of Science or Master of Science degrees) in economics (in addition to Bachelor of Arts or Master of Arts from Fine Arts or Humanities Departments).

Robert Solow [91] pointed out that, in the 1940s, economics had been basically a descriptive and institutional subject for a 'gentleman scholar'. The textbooks of those days were 'civilized' and discursive. ... "Formal analysis were minimal and it made economics the domain of intuitive economists". He concluded his summary of the state of economics near the end of the 20th century "with a paraphrase of Oscar Wilde's description of a fox hunt - 'the unspeakable in pursuit of the inedible'-saying that perhaps economics was an example of 'the over-educated in pursuit of the unknowable'." [91]. Despite the ongoing controversies today in the field of economics, the "New Millennium economists are far more comfortable with what they do after the changes in the structure and content of economics over the last half century" [92]. The root cause of these changes have been identified by Colander [92] to be due to the rise of Complexity Science since early 1980s. In fact, concepts from physics had continually been absorbed into the main stream economic formulation of ideas and models. As Venkat Venkatasubramanian noted in his recent book [93], "Concepts such as equilibrium, forces of supply and demand, and elasticity reveal influence of classical mechanics on economics. The analytical model of utility-based preferences can be traced back to Daniel Bernoulli, the great Swiss mathematical physicist from nineteenth century. One of the founders of neoclassical economics, Irving Fisher, was trained under the legendary Yale physicist, Jisiah Willard Gibbs, a co-founder of the discipline of statistical mechanics. Similarly, Jan Tinbergen, who shared the first Nobel Prize in Economics in 1969, was the doctoral student of the great physicist Paul Ehrenfest at Leiden University".

Indeed, more specifically as discussed in Section 2, we would like to correlate these changes to occur following the successful development in econophysics of the Simulated Annealing technique [22] in 1983 for Traveling Salesman type multi-variable optimization problems, and other successive developments in econophysics of analyzing correlations in stock prices (see, e.g., Reference [3,21]) or the kinetic exchange modelings of income and wealth distributions (see, e.g., References [28,63]). The statistical physics of TSP, as an example of successful developments in econophysics, had already been introduced in our 2010 econophysics textbook [32], which has been the only 'suggested textbook' (since inception in 2012) of the formal course on econophysics, offered (by Diego Garlaschelli) at the Physics Department of the Leiden University (see the course prospectus for 2012–2013 through that of 2020–2021 [94]), where one of the first Nobel-laureates in economics Jan Tinbergen came from.

Econophysics has come as an exceptional development in interdisciplinary sciences (see, e.g., Reference [95] for a popular exposition on this development). Historically, economics, more specifically social sciences, belonged to the Humanities departments and not of Science. For earlier interdisciplinary developments of Astrophysics, Biophysics, or Geophysics, the scenario and ambiance had been quite different. The mother departments had been parts of the same science schools and even the corresponding resources, like books, journals, and also the faculty, had strong overlaps and could be shared. The marriage negotiations for Econophysics have been difficult, though extremely desirable and natural; as the saying goes: "marriage between the King of natural sciences with the Queen of social sciences!"

Regular interactions and collaborations between the communities of natural scientists and social scientists are, however, rare, even today! Though, as mentioned already, interdisciplinary research papers on econophysics and sociophysics are regularly being published at a steady and healthy rate, and a number of universities (including Universities of Bern, Leiden, London, Paris, and Tufts University) are offering the interdisciplinary courses on econophysics and sociophysics, not many clearly designated professor positions, or other faculty positions for that matter, are available yet (except for econophysics in Universities of Leiden and London). Neither are there designated institutions on these interdisciplinary fields, nor separate departments or centers of studies for instance. Of course, there have been several positive and inspiring attempts and approaches from both economics and finance side (see, e.g., References [96,97], along with a number of those [66,67,98–100] from physics, which have already been appreciated in the literature). Indeed, the thesis [101] in August 2018, Department of History and Philosophy of Science, University of Cambridge, by financial economist Christophe Schinckus (one of the co-editors of this special issue), says that "In order to reconstruct the subfield of econophysics, I started with the group of the most influential authors in econophysics and tracked their papers in the literature using the Web of Science database of Thomson-Reuters (The sample is composed of: Eugene Stanley, Rosario Mantegna, Joseph McCauley, Jean-Pierre Bouchaud, Mauro Gallegati, Benoît Mandelbrot, Didier Sornette, Thomas Lux, Bikas Chakrabarti and Doyne Farmer). These key authors are often presented as the fathers of econophysics simply because they contributed significantly to its early definition and development. Because of their influential and seminal works, these scholars are actually the most quoted authors in econophysics. Having the 10 highest quoted fathers of econophysics as a sample sounds an acceptable approach to define bibliometrically the core of econophysics". In addition, the entry on 'Social Ontology' in The Stanford Encyclopedia of Philosophy [15], as discussed in Section 2, confirms positive impact of such econophysics and sociophysics research studies on the overall modern philosophical outlook of social sciences.

We may note, however, a recently published highly acclaimed massive (580 page) book [96] on economics ('landmark volume', said E. Roy Weintraub, 'creative, elegant and brilliant work', said W. Brian Arthur and 'written by master economists', said D. Colander) by (Late) Martin Shubik (Ex-Seymour Knox Professor Emeritus of Mathematical Institutional Economics, Yale University and Santa Fe Institute) and Eric Smith (Santa Fe Institute) discussed extensively on econophysics approaches and in general on the potential of interdisciplinary research studies inspired by the developments in natural sciences. Getting somewhat excited, I wrote to Martin Shubik in late 2016 that their book can also serve as an outstanding 'white-paper' document in favor of a possible Proposal for an International Center for Interdisciplinary Studies on Complexity in Social Sciences. He immediately responded and gave his impression about the difficulties involved and indicated very briefly about the minimal financial and structural requirements (both my letter to him and his response is appended below (Figures 11 and 12).

**Figure 11.** The first part of the email conversation between (late) Martin Shubik and BKC. Second part (email from BKC; appended to this part) is continued in Figure 12. The precise suggestions made in this immediate response indicate Shubik's prior plan for such 'interdisciplinary institutes' in economics.

**Figure 12.** Email conversation in the end of 2016 between (late) Martin Shubik and BKC regarding interdisciplinary developments in economics and the possibility of setting up an International Center for Interdisciplinary Studies on Complexity in Social Sciences. This email from BKC was appended to the response email (Figure 11) from Shubik. The (Yale) date and time mark in the mail-header (and that for BKC's in Figure 11, on arrival in Kolkata) indicate hardly any time gap between the two and the readiness with the precise suggestions indicate Shubik's prior thinking in similar line.

This ready and specific comments by Shubik clearly suggests that he actually had thought about the need of such an International Center for fostering interdisciplinary research which needs to be more inclusive than, for example, the Santa Fe Institute. The model of the Abdus Salam International Center for Theoretical Physics (ICTP), Trieste (funded by UNESCO and IAEA), was considered to provide helpful guidance for us here. It was contemplated, if an ICTP-type interdisciplinary research institute could be initiated for research studies on econophysics and sociophysics (see, e.g, Reference [102]). Though Shubik (who died in 2018 at the age of 92) agreed also to be one of its founding members, we could not make any progress yet. We may also note that Dirk Helbing and colleagues have been trying for an European Union funded 'Complex Techno-Socio- Economic Analysis Center' or 'Economic and Social Observatory' for the last decade or so (see Ref. [103] containing the White Papers arguing for their proposed project). We are also aware that Indian Statistical Institute had taken a decision to initiate a similar Center in India (see 'Concluding Remarks' in Reference [104]).

Hope, some such international visiting centers will come up soon and with them the spread of such interdisciplinary ideas will achieve more coherence and will lead to major success in such research studies.

**Author Contributions:** Data curation, A.S.; Writing—review & editing, B.K.C. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We acknowledge all our colleagues (mentioned by name in Section 2) for the collaborations. BKC is grateful to J.C. Bose National Fellowship (DST, Govt. of India) grant for support.

**Conflicts of Interest:** The authors declare no conflict of interest.


*3.3. Compressive Stress-Strain Curves*

The recorded stress–strain behavior of Group 1 specimens is shown in Figure 7. Figure 7a shows the stress–strain curves for the low concrete strength specimens in Group 1. It is evident that a substantial improvement in the peak compressive stress was observed. The important parameter to be observed is the range of strain for which the peak sustained stress was maintained. This suggests that LC-GFRP imparted a considerable ductility to the concrete, which is crucial for strengthening against dynamic loads. The post-peak behavior of Specimens SQ-LS-R0-2GFRP and SQ-LS-R0-4GFRP was descending, whereas a stable second branch was observed for Specimen SQ-LS-R0-6GFRP. On the contrary, the second branch of the compressive stress–strain curves of the high-strength specimens in Group 1 was descending irrespective of the number of LC-GFRP layers. The second difference in

*Sustainability* **2022**, *14*, x FOR PEER REVIEW 9 of 20

**Table 4.** Summary of the peak stress and the corresponding strain.

SQ-LS-R0-CON 8.66 0.707 - 0.0053 - SQ-LS-R0-2GFRP 15.87 4.243 83 0.0096 82 SQ-LS-R0-4GFRP 17.59 0.000 103 0.0155 194 SQ-LS-R0-6GFRP 20.51 1.414 137 0.0400 658 SQ-HS-R0-CON 14.41 4.950 - 0.0051 - SQ-HS-R0-2GFRP 21.62 4.243 50 0.0086 70 SQ-HS-R0-4GFRP 24.67 0.707 71 0.0131 159 SQ-HS-R0-6GFRP 26.48 2.121 84 0.0119 134 SQ-LS-R26-CON 7.74 0.707 - 0.0084 - SQ-LS-R26-2GFRP 16.71 1.414 116 0.0270 222 SQ-LS-R26-4GFRP 23.99 1.414 210 0.0500 495 SQ-LS-R26-6GFRP 29.29 8.485 278 0.0717 752 SQ-HS-R26-CON 13.39 2.121 - 0.0062 - SQ-HS-R26-2GFRP 23.16 1.414 73 0.0115 84 SQ-HS-R26-4GFRP 32.07 1.414 140 0.0290 364 SQ-HS-R26-6GFRP 40.73 6.364 204 0.0414 563

**Increase in Peak Stress (%)**

**Ultimate Strain**

**Increase in (%)**

**tion**

*3.3. Compressive Stress-Strain Curves*

**Specimen ID Peak Stress (MPa) Standard Devia-**

Figure 7a,b is the value of the ultimate strain. The high-strength specimens were able to sustain compressive stress to lower strain values as compared to low-strength specimens. were able to sustain compressive stress to lower strain values as compared to low-strength specimens.

The recorded stress–strain behavior of Group 1 specimens is shown in Figure 7. Figure 7a shows the stress–strain curves for the low concrete strength specimens in Group 1. It is evident that a substantial improvement in the peak compressive stress was observed. The important parameter to be observed is the range of strain for which the peak sustained stress was maintained. This suggests that LC-GFRP imparted a considerable ductility to the concrete, which is crucial for strengthening against dynamic loads. The post-peak behavior of Specimens SQ-LS-R0-2GFRP and SQ-LS-R0-4GFRP was descending, whereas a stable second branch was observed for Specimen SQ-LS-R0-6GFRP. On the contrary, the second branch of the compressive stress–strain curves of the high-strength specimens in Group 1 was descending irrespective of the number of LC-GFRP layers. The second difference in Figure 7a,b is the value of the ultimate strain. The high-strength specimens

**Figure 7.** Compressive stress vs. strain response for Group 1 specimens with (**a**) low concrete strength and (**b**) high concrete strength. Figure 8a,b shows stress–strain curves for Group 2 specimens with low and high concrete strength, respectively. A significant difference between Figures 7 and 8 is ob-

Figure 8a,b shows stress–strain curves for Group 2 specimens with low and high concrete strength, respectively. A significant difference between Figures 7 and 8 is observed in the second branch. It is recalled that the sharp corners in the Group 2 specimens were rounded to a 26 mm corner radius. The corresponding result is depicted in Figure 8a, as the stiff initial branch was followed by an ascending branch highlighting the importance of the corner radius. The peak compressive stress and the ultimate strain were found to increase with the number of LC-GFRP sheets. However, the improvement in the axial ductility was limited by the concrete strength. It is evident in Figure 8 that the failure of the high concrete strength specimens for the same number of LC-GFRP layers occurred at lower strains than those of the low concrete strength specimens. This could be related with the lower lateral dilation of the high-strength concrete as compared to the low-strength concrete. served in the second branch. It is recalled that the sharp corners in the Group 2 specimens were rounded to a 26 mm corner radius. The corresponding result is depicted in Figure 8a, as the stiff initial branch was followed by an ascending branch highlighting the importance of the corner radius. The peak compressive stress and the ultimate strain were found to increase with the number of LC-GFRP sheets. However, the improvement in the axial ductility was limited by the concrete strength. It is evident in Figure 8 that the failure of the high concrete strength specimens for the same number of LC-GFRP layers occurred at lower strains than those of the low concrete strength specimens. This could be related with the lower lateral dilation of the high-strength concrete as compared to the low-strength concrete.

**Figure 8.** Compressive stress vs. strain response for Group 2 specimens with (**a**) low concrete strength and (**b**) high concrete strength. **Figure 8.** Compressive stress vs. strain response for Group 2 specimens with (**a**) low concrete strength and (**b**) high concrete strength.

The effect of the concrete strength on the gain in peak compressive stress due to LC-GFRP confinement is shown in Figure 9. It is evident that the gain in peak compres-

number of LC-GFRP layers, the specimens with low concrete strength demonstrated greater improvement in the peak compressive stress than high strength specimens. Another observation that can be made from Figure 9 is the effect of corner radius on the improvement in peak compressive stress. For the case of no corner radius (see Figure 9a), the maximum increase in the peak compressive stress observed for Specimen SQ-LS-R0-6GFRP was 137%, whereas the corresponding value for a 26 mm corner radius

was 278%, observed for Specimen SQ-LS-R26-6GFRP.

*3.4. Effect of Concrete Strength and Corner Radius*


**HUNTINGTON'S DISEASE - MOLECULAR PATHOGENESIS AND CURRENT MODELS**

Edited by **Nagehan Ersoy Tunalı**

#### **Huntington's Disease - Molecular Pathogenesis and Current Models**

http://dx.doi.org/10.5772/62999 Edited by Nagehan Ersoy Tunalı

#### **Contributors**

Stéphanie Tomé, Elodie Dandelot, Xavier Xifro, Marta Anglada, Núria Cabezas, Laura Vidal-Llobet, Jordi Alberch, Sophie Precious, Zdenka Ellederova, Petra Rausova, Petra Vochozkova, Daniela Vidinska, Eva Hrnciarova, Bozena Bohuslavova, Monika Macakova, Ivona Valekova, Stefan Juhas, Taras Ardan, Petr Solc, Jan Motlik, Irina Kerkis, Cristiane Valverde Wenceslau, Nagehan Ersoy Tunalı

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First published in Croatia, 2017 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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Effect of Textures and Microstructures on the Occurrence of Delamination during and after Fracture Toughness Tests of API X80 Steel Plates

*Fabio Faria Conde, Haroldo Cavalcanti Pinto, Mohammad Masoumi and Julian Arnaldo Avila*


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**2**

*Brazil*

**The Mystery of P2X7 Ionotropic Receptor:**

Ion channels are aqueous pores which allow ions to pass based upon an electrochemical gradient. In order to conduct ions, these channels modify their shape by a process called gating, a shifting between opening and closing states. These channels are gated by various stimuli i.e. applied electric field, chemical transmitters, pH, heat and cold, among others. When an electrical potential is held constant in a cell, the gating process equilibriates rather than reaching an energy-dissipating position (a cyclic steady state). Ions diffuse through open channels very fast, involving an interaction between ions, the pore, and solvent that lead to ionic selectivity, saturation, block and flux coupling (Hille, 1975a;

The ion channel has generally been viewed as having unchanging selectivity strengthening the argument that there is a degree of specificity between ion and channel. Nevertheless, some classes of ion channels present a more varied selectivity of ions, allowing impermeable ions or molecules to permeate under certain circumstances. However, the there is no a systematic approach to distinguish different ion channels and in most cases, it is necessary to differentiate the channels by their kinetics, molecular sequence, pharmacological

The diversity of channels continues to pose numerous areas to explore in physiology, molecular biology, pharmacology and biophysics.(Hille, 1991). Ion channels are generally classified into two groups: the channels that allow the passage of ions (Na+, K+, Ca2+) and those that allow the passage of large ions and solutes (ie ATP, glutamate, fluorescent dyes of low molecular weight). This second group is predominantly made up of large conductance ion channels which when activated change the permeability of cell membranes for molecules of up to 1000 Daltons (Ojcius, D. M. & Ding-E Young, J., 1990; Iacovache et al, 2010). These channels are and allow for the secretion of ATP and other intracellular

molecules, not to mention implicated in various mechanisms of cell death.

**1. Introduction 1.1 Ion channels**

Läuger, 1973a).

properties, and response to ionic substitution.

**From a Small Conductance Channel**

**to a Large Conductance Channel**

R.X. Faria, L.G.B. Ferreira and L.A. Alves

*Laboratory of Cellular Communication,*

*Oswaldo Cruz Institute, Oswaldo Cruz Foundation,*