**Acknowledgements**

*New Innovations in Engineering Education and Naval Engineering*

of the mundane tasks in: course creation (*by instructors*), course management (*by LMS's*), course participation (students), grading and course evaluation methodology, and issuing valid certification. The machine age that will make our transition to a fully digital society brings with it a whole slew of new realities as well for the professional. The expectations from each of us has become compounded to an extent that a degree certificate attached to an individual that requires skills are also

Professional degree certificates (*undergraduate and graduate*) now also comes with it the responsibilities of skills upgrading on a continuing basis; of being in communities attached to that profession; of being in the know in those communities; of being socialized in the platforms of Facebook, Twitter, Instagram, and LinkedIn to not just being there but being a part of the larger whole comprised of billions of people in the world. To think about it in terms of how AI responds to that changed perspective is to assume that all these interactions are captured by the AI machine and adds it in terms of quantifiable data, making new assumptions about the added data in effect, understanding ourselves more from that newly captured information producing an updated profile of a person's learners style based on captured cognitive functions and user action decisions as opposed to self-reported learner style [22]. Schneider [21] points out that through data analytics, these learner characteristics can be extracted automatically from user's ongoing interaction to perform a variety of transactions (*from finding a route using GPS* (*geographical positioning system) or learning online to online banking transaction*) in day-to-day lives. When these patterns of user interaction are validated, a method called "user nudging" could be used much like adaptive computing that "adjusts to serving user tasks based on prior action," that is,

updated be it technical, socialization, community, or connectivity [21].

nudging would guide user actions by giving choices through prompts.

**8.1 Security concerns with respect to PLErify, MOOC, and tech tools**

Denning [23] summarizes it brilliantly that to truly survive in the age of machines

Security breaches from China, North/South Korea, and Russia are a threat to our tech-enabled life. These countries' very advanced cyber-surveillance and intrusion system have penetrated US cyber defense system potentially undoing major education technology advancements. The industry needs to come up with a very strong authentication system as well as cyber-blocking mechanisms beyond the obvious

Without a strong cyber security strategy attached to all these tech innovations, any attempts at technologizing higher education would face enormous challenges. China's breaches covered the entire hardware/software and telecommunication ecosystem (home routers included) baffling Europe, the US, and Australia. A solution that has been proposed is virtualization and containerization. If virtualization and containerization provides a guarantee for the safety and security of cumulative progress and strides made in the education sector and if we are willing to adapt to rapid changes demanded of us as educators, faculty, and students, then the future

where the knowledge worker conducts work on highly intelligent machines, new expectations come to the fore that requires pragmatism in belongingness, ever adapting skillsets that changes as the system changes, community building based on chosen areas of belongingness (professional, leisure, or recreational), and last but not least, willingness to mentor, to display your skills to the person that needs it so that the next learner improves the knowledge to the next and so on and so forth.

**62**

will certainly be bright.

**8. Conclusion**

firewalls.

After incorporating additional critique (style, formatting, and punctuation) from my children who are both writers in their own right, I finally produced my chapter contribution to the book 2019 Engineering Design and Innovation Methods at IntechOpen. I take this opportunity to convey my deep heartfelt appreciation to IntechOpen's generous partial publication waiver which assisted me in pursuing further publication of my manuscript with them. Thank you.

For being my constant inspiration and for whom I am constantly reminded that we must perform our responsibility as trustful stewards of technology for the future of the young generation and the forthcoming ones thereafter, I dedicate this work to my children Avinash A. Kunnath (AB Mathematics, University of California Berkeley, 2008) and Ameeta A. Kunnath (BEngg Structures, University of California San Diego, 2015) who, along with members of their generation, will continue the work in their chosen careers and professions to improve various facets of our digital society we are all part of as technology continuously shapes and impacts our modern lives by the minute.


**2. Nanomedicine**

Nowadays, nanomedicine is an emerging and highly relevant area due to the fact that great advances have been made in the treatment of various diseases, such as cancer, neurodegenerative and cardiovascular diseases, and hormonal problems. To understand the development and possible applications of nanomedicine, it is necessary to define the concept of nanotechnology.


**3. Discussion**

We considered cytotoxicity of any AM monomer to be a major determinant of whether the materials that incorporate the monomer in their resin phase are worthy of further study. We believe that direct contact cellular testing of the new agent must be done at biologically relevant eluent concentrations. In this study, we employed conditions that reflect the accelerated leachability of UPH resins and included AMsil concentrations that significantly exceed the upper thresholds established experimentally in the previous work [45]. The direct toxicity of AMsils towards CCL1 cells and/or HGFs was demonstrated to be marginal or undetectable, except at the higher concentrations of monomers tested. These high AMsil levels correspond to unrealistically high levels of the unreacted monomers and are highly unlikely to ever be registered clinically. Our cytotoxicity results support the basic hypothesis of the study and suggest that, from a biotoxicity viewpoint, AMsils can be safely utilized in design of AM new materials. Leachability studies of AM–UPE formulations employing high-performance liquid chromatography are currently underway in our laboratory, and are expected to confirm the conclusions derived from the tests based on the accelerated UPH leachability study.

Upon introduction of AMsil into the UPE resin, a shift towards lower CA values, consistent with the moderate increase in the overall hydrophilicity, was seen in all AMsil–UPE formulations. The detected range of CAs in AMsil–UPE resins (37.4–53.3◦) correlates very well with the range of CAs typical for the commercial resin composites (37.4–53.3◦) [46]. The lowest CAs detected in 10% AMsil2–UPE formulation make this resin a good candidate for the incorporation of the ACP filler in future design of AMRE composites. ACP-filled composite requires sufficient water absorption to initiate water-catalyzed transformation of ACP during which the remineralizing calcium and phosphate ions are released, by diffusion, into surrounding mineral-deficient tooth structures. There, they regenerate these mineral-depleted structures via redeposition of hydroxyapatite [47]. Taken together, the enhanced wettability should ease a diffusion of water into AMRE composite and result in the subsequent release of calcium and phosphate ions from the composite needed for demineralization prevention and/or active remineralization at the restoration site.

The range of DVC values attained in AMsil–UPE copolymers (60.7–86.7%), dependent on both the monomer type and its quantity in the resin, were higher or equal to the DVC reported for 2,2-bis[p-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bis-GMA)-based resins/composites with incorporated QA ionic dimethacrylate (67.9–70.7%) [25]. AMsil2 copolymers reached significantly higher DVC values (75.2–86.7%) compared to their AMsil1 counterparts (60.7–70.9%). In AMsil2–UPE

formulations, the inclusion of AM monomer apparently did not a ffect the high levels of DVC typically seen in UDMA-based resins [35,36]. This phenomenon has been attributed to the chain transfer reactions caused by UDMA's –NH– groups, resulting in increased mobility of the resin network's radical sites [48]. The DVCs attained in AMsil2–UPE copolymers suggest limited mobility of cross-linked polymer matrix, thus reducing the likelihood of unreacted monomers leaching out to a minimum. The observed DVC decrease in AMsil1–UPE formulations compared to those of AMsil2–UPE is yet to be explained.

The results of FS and E tests indicated a reduction of the copolymers' mechanical properties in going from the UPE control to 10% AMsil–UPE to 20% AMsil–UPE. The reduction was far more pronounced in AMsil1 series (50%) compared to the AMsil2 series (15%). This overall reduction in mechanical properties, particularly in AMsil2–UPE copolymers, should not disqualify this monomer from further exploration, as AM agent in multifunctional AMRE composites. To compensate for the reduction in the mechanical properties, incorporation into composites of the reinforcing fillers in addition to ACP should be considered.

AMsils integrated into UPE resin were e ffective in reducing (*p* ≤ 0.002) *S. mutans* biofilms. Compared to the commercial control, *P. gingivalis* biofilm biomass was notably lower (71–85%) on AMsils–UPE copolymer disks, however, not statistically di fferent. As Gram-positive bacteria have peptidoglycan with long anionic polymers, called teichoic acids [49] (i.e., yielding a higher cell surface net negative charge than Gram-negative organisms), one could anticipate *S. mutans* to be more susceptible to AMsils. Notwithstanding, quaternary ammonium compound AM functionality can also be a ffected by the type of counter-ion [50], pendant active groups [51], molecular weight, and length of the alkyl chains [52].

AMsils show promise, as *S. mutans* planktonic and biofilm forms were reduced. Nonetheless, this reduction was only significant (*p* ≤ 0.002) for biofilms. When comparing planktonic and biofilm responses, similar trends were observed with an endodontic sealer. For example, bacteria tested with a resin-based root canal sealer did not statistically reduce the planktonic forms, while notably decreasing bacteria in monospecies biofilms [53]. These authors attributed this to the release of substances during the setting process. This attribute is unlikely applicable to our material, as DVC was high and aqueous extraction was conducted for 72 h. In another report, *Staphylococcus aureus* biofilms were demonstrated to be more susceptible to killing than the planktonic form of the same strain [54].

Although AM functionality is observed, we believe that the full potential of the AMsil monomers has not yet been realized. As reported for other quaternary ammonium compounds [47], the current AMsil–UPE copolymer formulations are likely to have N+ charges randomly distributed throughout the material. As the mechanism of AM action is contingent on contact, it would be advantageous to develop fabrication methods that would favor charge density at the materials surface. Further, others have demonstrated that proteins can diminish the AM capability of quaternary ammonium methacrylates (reviewed by [6]). Currently, there is insu fficient information concerning the interaction of proteins with quaternary ammonium methacrylates. Elucidation of the protein–material interactions would yield valuable information to develop strategies to maximize AM e fficacy of materials with a charge-based AM mechanism of action.

In conclusion, our novel AM dental monomers (AMsil1 and AMsil2) exhibited minimal or no toxicity upon direct contact with biologically relevant concentrations, while reducing *S. mutans* and *P. gingivalis* biofilm forms. AMsils made the UDMA/PEG-U/EHMA resin more hydrophilic. This would be an advantageous feature in AMRE composites that require water to induce their remineralizing e ffects. At 10 mass % level of AMsil monomer, DVC of the ensuing AMsil–UPE copolymers was only marginally lower than in UPE control and still exceeded DVCs typically seen in the commercial composites based on bis-GMA/triethyleneglycol dimethacryalate (TEGDMA) resins. The mechanical properties of AMsil–UPE copolymers were reduced (11–57%) compared with the UPE control. The extent of reduction depended on both the type and the concentration of AMsil monomer in the resin. This finding should

not disqualify the AMsil–UPE resins from use in AMRE composites intended for Class V restorations where the mechanical stability is not a critical factor.

#### **4. Materials and Methods**

## *4.1. Monomer Synthesis*

The synthesis and validation protocols for the AMsils are described in detail by Okeke et al. (2019) [40]. In brief, AMsil1 and AMsil2 were synthesized at 50–55 ◦C by reacting equimolar amounts of tertiary amine, DMAEMA, with IPTMS and BrUDTMS, respectively, in the presence of chloroform and butylated hydroxytoluene. DMAEMA, IPTMS, and butylated hydroxytoluene were purchased from Sigma, St. Louis, MO, USA. BrUDTMS was purchased from Gelest Inc., Morrisville, PA, USA. Reactants and solvents (chloroform, diethyl ether, hexane; Sigma, St. Louis, MO, USA) used during synthesis and the subsequent purification were used as received, without further purification. The reaction yields were 94.8% and 36.0% for AMsil1 and AMsil2, respectively. Due to the generally hygroscopic nature of QA monomers, the AMsils were stored under vacuum (25 mm Hg) before being used for resin formulation and/or copolymer disk specimen preparation.

## *4.2. Structural Verification*

Purified monomers were characterized by 1H and 13C NMR spectroscopy as described [40]. Briefly, spectra were obtained using a Bruker Advance II (600 MHz) spectrometer equipped with a Broadband Observe room temperature probe (Bruker, Corp., Billerica, MA, USA). Monomers were dissolved in deuterated dimethyl sulfoxide containing tetramethylsilane.

#### *4.3. Experimental Resin Formulation*

UPE resin was formulated from the commercially available monomers UDMA, PEG-U, and EHMA at 2.8:1.0:1.7 mass ratio (corresponds to the average mass ratio of the ternary UDMA-based formulations explored by our group so far). A conventional visible light initiator system comprised of camphorquinone and ethyl-4- *N*,*N*-dimethylamino benzoate (4EDMAB) was introduced to the resin at concentrations of 0.2 mass % camphorquinone and 0.8 mass % 4EDMAB. AMsils were blended into UPE resin to yield (AMsil1 or AMsil2)–UPE resin with 10 or 20 mass % of AM component. Addition of AMsil1 or AMsil2 to the light-activated UPE resin took place in the absence of blue light. The rationale for the chosen levels of AM monomers is based on the previously reported AM activities of similar QA methacrylates in camphorquinone /4EDMAB-activated bis-GMA/TEGDMA resins [25]. Once all components were introduced, the mixture was stirred magnetically (38 rad/s) at 22 ◦C until a uniform consistency was achieved. CLEARFIL SE Protect BOND (Kurary America, Inc., New York, NY, USA) was used as a comparative commercial AM material. This resin was prepared using equal quantities of primer (containing MDPB) and bonding (containing bis-GMA-HEMA) agent.

## *4.4. Biocompatibility Tests*

Direct contact cytotoxicity of AMsils was determined following described protocols [55,56]. Briefly, immortalized mouse subcutaneous connective tissue fibroblasts (NCTC clone 929 [L-cell, L-929, Strain L derivative]; American Type Culture Collection (ATCC), Manassas, VA, USA) (CCL1) or HGF (Applied Biological Materials, Inc., Richmond, BC, Canada) were exposed to 2-fold serial dilutions (AMsil1: ≤8.34 mmol/L; AMsil2: ≤7.28 mmol/L). Chosen concentrations corresponded to approx. 7% mass fraction of AMsil1 or AMsil2 in the copolymer resin and a maximum of 2% leaching. To allow for the possibility of restoration multiplicity and variable size, a 2-fold greater dilution was also included in the testing. These calculations are based on the accelerated leachability study of UDMA/PEG-U/2-hydroxyethyl methacrylate (HEMA) resin (abbreviated UPH; a close analog to UPE resin used in this study) and ACP-UPH composites [57]. After 24 and 72 h incubation, cells were assessed for cell viability (LIVE/DEAD ® Viability/Cytotoxicity kit, Life Technologies, Corp., Grand

Island, NY, USA) and metabolic activity (CellTiter ® AQueous One Solution Reagent; Promega, Corp., Madison, WI, USA). Controls were without the AMsils and/or cells. The CCL1 cells and HGFs were maintained, at 37 ◦C and 5% CO2, in 10% serum-supplemented Eagle's minimum essential medium (ATCC) and PriGrow III medium (Applied Biological Materials, Inc.), respectively. For experiments, cells were obtained from a subconfluent stock culture. Means were obtained from 5 independent replicates tested in duplicate.

#### *4.5. Contact Angle (CA)*

Changes in hydrophilicity/hydrophobicity of UPE resins due to the introduction of AMsils were assessed by CA measurements (drop shape analyzer DSA100, Krüss GmbH, Hamburg, Germany). Following the deposition of the sessile droplets of the resin on the substrate, they were imaged after 1 min resting time with a charge-coupled device camera at the points of intersection (three-phase contact points) between the drop contour and the projection of the surface (baseline). The CA water values were calculated employing the Krüss Advance software. Four repetitive measurements were performed in each group.

#### *4.6. Copolymer Specimen Preparation*

For biotesting, UPE and AMsil1–UPE and AMsil2–UPE copolymer specimens were fabricated by filling circular openings of a flat stainless-steel molds (6 mm diameter, 0.5 mm thickness) with the resins. Each side of the mold was covered with Mylar film and a glass slide, firmly clamped, and then cured (2 min/side: Triad 2000; Dentsply International, York, PA, USA).

Specimens were subjected to extraction in Dulbecco's phosphate-bu ffered saline lacking both calcium and magnesium (Life Technologies, Grand Island, NY, USA) for 72 h, at 37 ◦C and 6.3 rad/s using an orbital shaker. After extraction, the disks were dried under vacuum (desiccator; ~22 ◦C) for 7 days. Specimens were sterilized for 12 h using an Anprolene gas sterilization chamber (Andersen Products, Inc., Haw River, NC, USA). Prior to bacterial testing, specimens were degassed for ≥5 days under vacuum (desiccator; ~22 ◦C).

#### *4.7. Degree of Vinyl Conversion (DVC)*

DVC of UPE and (AMsil1 or AMsil2)–UPE resins was determined by collecting the near-IR (NIR) spectra (Nexus; ThermoFisher, Madison, WI, USA) before and 24 h after the light cure and calculating the reduction in =C–H absorption band at 6165 cm−1 in the overtone region in going from monomers to polymers. By maintaining a constant specimen thickness, a need for an invariant internal standard was eliminated. The DVC was calculated as

$$\text{DVC (\%)} = \text{[(area}\_{\text{mononner}} - \text{area}\_{\text{polymer}}) / \text{area}\_{\text{mononner}}] \times 100 \tag{1}$$

where areapolymer and areamonomer correspond to the areas under 6165−1 absorption peak after and before the polymerization, respectively.

#### *4.8. Mechanical Properties of Copolymers*

Test specimens (2 mm × 2 mm × 25 mm) for flexural strength (FS) and elastic modulus (E) determinations were photopolymerized in the same manner as the copolymer disks for biological testing. Polymerized specimens did not undergo any additional treatment. The FS and E of UPE and (AMsil1 or AMsil2) UPE copolymer specimens was tested employing the Universal Testing Machine (Instron 5500R, Instron Corp., Canton, MA, USA). The load was applied (crosshead speed of 1 mm/min) to the center of a specimen positioned on a test device with supports 20 mm apart. The FS and E of the specimens (three replicates/experimental group) were calculated as instructed in the ISO4049:2009 document.

#### *4.9. Bacterial Testing*


**5. Apo-B100 is a flexible string wrapped around the surface of LDL**

As already indicated above, the physicochemical state of the core lipids is intimately related to the structure and dynamics of the particle surface, which consists of about 700 phospholipid molecules and one single copy of apo-B100. Apo-B100 is a huge glycoprotein and its polypeptide chain consists of 4536 amino acid residues with an estimated molecular mass of about 550 kDa for the glycosilated form [52,53]. Apo-B100 is a single chain protein with a total contour length of about 70 nm [54] and can be viewed as a highly flexible molecular string composed of single domains [20]. Five consecutive domains were identified based on secondary structure elements representing the main conformational motifs of apo-B100. The single domains are connected by flexible interdomain linker regions, which allow relative movements of domains to each other. The feasibility of such motions was shown in a low resolution model of detergent solubilized apo-B100, which was derived from small angle neutron scattering data [55]. In this model, compact rigid domains are visible being connected by flexible interdomain linkages, which possess a substantial degree of freedom in their spatial orientation. A hypothetical spatial arrangement of the apo-B100 molecule on a spherical LDL particle was created after assigning the secondary structure elements, which were deduced from a secondary structure prediction, to the surface of apo-B100 (Figure 4). Likewise, the averaged surface shape of the 3D-model would allow for variations in the thickness of the apo-B100 molecule by about 1 nm. Such variations are most likely required to compensate for changes in the surface area upon lipid exchange and particle shrinking during endogeneous lipoprotein conversion from very low density lipoprotein (VLDL) to LDL.

**Figure 4.** Reconstituted low resolution model of lipid-free apo-B100 derived from small angle neutron scattering data. Apo-B100 shows an elongated arch-like morphology indicating single domains and mobile less defined linker regions. A hypothetical model of a spherical LDL particle after superposition of the structural model of apo-B100 is shown (adapted from ref. [55]). Secondary structure modules are assigned to the surface after a secondary structure prediction was performed. The results nicely correspond to the pentapartite model suggested by [20].

Concerning the topology of apo-B100 on the surface of LDL the most detailed information is obtained from cryo-e.m. images (see also Figure 2). Chatterton et al. were among the first to visualize apo-B100 as string circumventing LDL, and to report on mapped epitopes of apo-B100 distributed over one hemisphere of the LDL particle [56,57]. Recent single particle 3Dreconstructions from immuno cryo e.m. images delineated a more accurate picture of apo-B100 revealing a looped topology of the protein backbone with distinct epitopes identified along the protein chain. According to this model, epitopes in the LDL receptor binding domain are located on one side of LDL, whereas epitopes located in the N-terminal and Cterminal domains are in close vicinity to each other on the opposite side of LDL [36]. In addition, a prominent protrusion is visible in the images at the pointed end of the particle. A similar knob-like region was apparent in the low resolution model of lipid-free apo-B100 shown in Figure 4. This protrusion most probable represents the non-lipid associated globular N-terminal domain of apo-B100, which shows a high homology to lamprey lipovitellin [58]. Except for the N-terminal domain, little is known about the molecular organisation of the structural motifs, whose amphipathic nature determine lipid association. However, to evaluate lipid-protein interactions physical parameter like interfacial elasticity or molecular dynamics have to be considered. In this context, it was suggested that the hydrophobic β-sheet domains of apo-B100 act as elastic lipid anchors, whereas the amphipathic α-helical domains respond rapidly to changes in surface pressure [59,60]. In any case, it can be assumed that alterations in the adsorption and penetration depth of apo-B100 in the phospholipid monolayer and in the lipid core are accompanied by structural rearrangements of the domains and changes in the orientation of the domains relative to each other. In the course of such elastic motions, intramolecular rearrangements are likely to alter the overall hydrophobicity and surface activity of single protein domains. These modifications not only affect lipid-protein interaction, but are equally important for molecular and cellular recognition of apo-B100.

10 Lipoproteins – Role in Health and Diseases

susceptibility of LDL particles to oxidative modifications and lipid peroxidation might be correlated to temperature [18]. As oxidized LDL play a crucial role in the pathogenesis of atherosclerosis, any contribution to the comprehension of antioxidant efficiency may be of therapeutic potential [2,51], further pointing to the physiological relevance of the lipid core

As already indicated above, the physicochemical state of the core lipids is intimately related to the structure and dynamics of the particle surface, which consists of about 700 phospholipid molecules and one single copy of apo-B100. Apo-B100 is a huge glycoprotein and its polypeptide chain consists of 4536 amino acid residues with an estimated molecular mass of about 550 kDa for the glycosilated form [52,53]. Apo-B100 is a single chain protein with a total contour length of about 70 nm [54] and can be viewed as a highly flexible molecular string composed of single domains [20]. Five consecutive domains were identified based on secondary structure elements representing the main conformational motifs of apo-B100. The single domains are connected by flexible interdomain linker regions, which allow relative movements of domains to each other. The feasibility of such motions was shown in a low resolution model of detergent solubilized apo-B100, which was derived from small angle neutron scattering data [55]. In this model, compact rigid domains are visible being connected by flexible interdomain linkages, which possess a substantial degree of freedom in their spatial orientation. A hypothetical spatial arrangement of the apo-B100 molecule on a spherical LDL particle was created after assigning the secondary structure elements, which were deduced from a secondary structure prediction, to the surface of apo-B100 (Figure 4). Likewise, the averaged surface shape of the 3D-model would allow for variations in the thickness of the apo-B100 molecule by about 1 nm. Such variations are most likely required to compensate for changes in the surface area upon lipid exchange and particle shrinking during endogeneous

**5. Apo-B100 is a flexible string wrapped around the surface of LDL**

organisation. However, this vital question still remains unanswered.

lipoprotein conversion from very low density lipoprotein (VLDL) to LDL.

correspond to the pentapartite model suggested by [20].

**Figure 4.** Reconstituted low resolution model of lipid-free apo-B100 derived from small angle neutron scattering data. Apo-B100 shows an elongated arch-like morphology indicating single domains and mobile less defined linker regions. A hypothetical model of a spherical LDL particle after superposition of the structural model of apo-B100 is shown (adapted from ref. [55]). Secondary structure modules are assigned to the surface after a secondary structure prediction was performed. The results nicely

#### **6. Apo-B100 containing lipoproteins are very soft and flexible**

LDL particles are formed in the circulation by lipolytic conversion of TG-rich VLDL particles. This enzyme mediated endogenous transformation is accompanied by an extensive shrinking in particle size from about 50-80 nm for VLDL to ~20 nm for LDL. In the course of remodelling, apo-B100 remains bound to its nanocarrier stabilizing the lipid assembly by maintaining structural integrity. To accomplish this, apoB100 has to become more condensed or relaxed depending on the lipid packing density. Likewise, this dynamic process is modulated by the molecular mobility of the surrounding microenvironment. To test for this hypothesis we have recorded temperature dependent molecular motions in VLDL and LDL particles using elastic incoherent neutron scattering [61]. With this technique, motions in the nano- to picosecond time scale can be recorded. The calculated dynamic force constants are a direct measure for the resilience of the particles. The results show that at physiological temperatures VLDL particles are very soft, elastic and mobile as compared to LDL, which is more rigid (see Figure 5). This observation supports the notion that apo-B100 in VLDL is loosely packed at the interface covering a large surface area with

low interfacial surface tension [59]. During particle conversion from VLDL to LDL, however, the relative number of surface molecules increases and a higher molecular packing density leads to a compression of the lipid anchored protein regions and an overall stiffening of the LDL particle [60].

**Figure 5.** Molecular motions in LDL and VLDL. Elastic temperature scans are recorded with elastic incoherent neutron scattering. The mean square thermal fluctuations (<u2>) are shown as function of temperature. The molecular resiliences are derived from the slopes in the curves. It is seen that VLDL has an increased motion at elevated temperatures compared to LDL. Parts of this figure are reproduced, with permission, from ref. [60].

To conclude, the intrinsic conformational flexibility and elasticity of apo-B100 containing lipoprotein particles is most likely critical for specific affinities of lipoproteins to receptors, antibodies or enzymes. Moreover, it would seem that the susceptibility of lipoproteins to oxidative modifications and hence their atherogenicity is influenced by their dynamic nature.

### **7. LDLs are flexible nanotransporters circulating in blood**

In the search for new and improved therapeutics, the field of nanomedicine dealing with functionalized nanoparticles for molecular imaging and therapy is rapidly emerging. Nanoparticles offer new opportunities to transfer active substances directly to the diseased site in the body. By additional surface coatings or functionalizations, the properties of nanoparticles can be tuned to specific needs. Within the last two decades, a variety of artificial nanoparticles have been designed for targeted delivery of drugs or contrast agents. Many of these nanoconstructs are developed for cancer therapy taking advantage of the leaky vasculature of tumours. Apart from tumour targeting, increasing efforts are devoted to the treatment and imaging of atherosclerotic plaques (for a recent review see ref. [62]). Over time, a broad and versatile nanoparticle platform was created in which liposomes and biodegradable polymers have turned out to be the most promising candidates. It is important to mention that several nanomedicine products have already been established on the market and numerous products are successfully applied in clinical trials [63]. However, inherent problems of nanoparticles are biocompatibility and low stability in vivo, since most nanoparticles become rapidly cleared by the reticuloendothelial system. In contrast to artificial systems, lipoproteins are naturally occurring nanoparticles evading recognition by the body´s immune system. Hence, lipoproteins are excellent candidates with attractive properties to be considered as molecular transporters. A great advantage of LDL over other nanoparticles is the fact that LDL particles stay in circulation for several days, and are not cleared immediately by the mononuclear phagocyte system of the liver and spleen. The average lifetime of an LDL particle is 2-3 days and this time span is about three times longer as reported for long-circulating liposomes, currently applied in chemotherapy [64]. It was recognized that certain tumor cells overexpress LDL receptor, however, the targeting specificity is limited as the LDL receptor is ubiquitously expressed throughout the body, most prominent in the liver. However, using apo-B100 as inherent targeting sequence the enhanced circulation times in blood enable drug-loaded LDL particles to bind to specific receptors exposed on the surface of e.g. tumor or atherosclerotic plaque. Once recognized by the receptor, the functionalized LDL particles become internalized, accumulate in the tissue and exert an enhanced effect (reviewed by [65]). The intrinsic targeting properties of LDL to atherosclerotic plaques are already utilized for early diagnosis and detection of atherosclerotic lesions by different imaging modalities (for reviews see refs. [66,67]). However, to modify lipoprotein particles for medical purposes, care has to be taken not to compromise essential biophysical and structural features of LDL with the goal to preserve the biological activity. In general, there are several possibilities to create multifunctionalized lipoprotein particles. Some representative examples are shown in the scheme in Figure 7. One possibility is to load hydrophobic drugs (e.g. chemotherapeutics, antibiotics, vitamins, signal emitting molecules or small nanocrystals) in the lipophilic inner core of LDL. This can be accomplished by different techniques including lyophilisation, solvent evaporation and reconstitution procedures [68,69]. However, LDL particles can not be reconstituted so easily and remote drug/contrast agent loading into native lipoprotein particles is still a tedious approach currently not being standardized. Amphiphilic substances (drugs or marker molecules) or fatty acid modified chelator complexes can be incorporated in the PL monolayer [70,71]. This has successfully been done in numerous biophysical studies and for diagnostic purposes. Finally, the surface of LDL can be modified by protein labeling. This is done by covalent attachment of substances to the lysine and cysteine amino acid residues of apo-B100. Such substances include fluorophores, radionuclides or metal ions for molecular imaging [65]. Alternatively, targeting sequences (e.g. folic acid) can be coupled to apo-B100 with the purpose to reroute LDL to alternate receptors, which, in case of folate, are more specifically expressed in tumor cells [72].

12 Lipoproteins – Role in Health and Diseases

with permission, from ref. [60].

nature.

LDL particle [60].

low interfacial surface tension [59]. During particle conversion from VLDL to LDL, however, the relative number of surface molecules increases and a higher molecular packing density leads to a compression of the lipid anchored protein regions and an overall stiffening of the

**Figure 5.** Molecular motions in LDL and VLDL. Elastic temperature scans are recorded with elastic incoherent neutron scattering. The mean square thermal fluctuations (<u2>) are shown as function of temperature. The molecular resiliences are derived from the slopes in the curves. It is seen that VLDL has an increased motion at elevated temperatures compared to LDL. Parts of this figure are reproduced,

To conclude, the intrinsic conformational flexibility and elasticity of apo-B100 containing lipoprotein particles is most likely critical for specific affinities of lipoproteins to receptors, antibodies or enzymes. Moreover, it would seem that the susceptibility of lipoproteins to oxidative modifications and hence their atherogenicity is influenced by their dynamic

In the search for new and improved therapeutics, the field of nanomedicine dealing with functionalized nanoparticles for molecular imaging and therapy is rapidly emerging. Nanoparticles offer new opportunities to transfer active substances directly to the diseased site in the body. By additional surface coatings or functionalizations, the properties of nanoparticles can be tuned to specific needs. Within the last two decades, a variety of artificial nanoparticles have been designed for targeted delivery of drugs or contrast agents. Many of these nanoconstructs are developed for cancer therapy taking advantage of the

**7. LDLs are flexible nanotransporters circulating in blood**

**Figure 6.** Scheme giving some examples of how LDL particles can be modified to act as natural endogenous nanoparticles for targeted drug delivery or multifunctional molecular imaging.

To construct lipoprotein mimetic particles, also referred to as lipoprotein related particles, artificial lipoprotein particles have to be reassembled from individual lipid and protein entities. This approach was highly successful for high density lipoproteins using apo-AI mimetic peptide sequences [73]. For LDL, this approach was not pursued yet and will be much more complicated considering the complex dynamic nature of apo-B100.

Over the last few years, a promising nanoparticle platform was established, which exploits the endogenous properties of natural lipoproteins being non-toxic, non-immunogenic and biodegradable. Although this platform still offers vast potential for improvements, first promising results in enhanced multimodal imaging of tumors and atherosclerotic plaques are achieved giving hope that further endeavors to combine diagnostics and personalized therapeutics will also be successful.


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## Meet the editors

Dr. Muhammad Imran received his Ph. D from the University of Agriculture, Faisalabad in 2013 with distinction. His research interests are the preparation of cost-effective polyphenol based dietary interventions against metabolic syndromes and conversion of agro-waste materials into value-added products along with expertise in functional and nutraceuticals foods. He has more than 85 publications in impacted international and nation-

al journals with a cumulative impact factor of more than 200. He has also published 11 book chapters with international publishers such as Springer and Elsevier. Moreover, he is also working as section head of a Research Unit and has produced more than 14 M. Phil students within his research area. He has also served as a guest editor for international journals.

Dr. Ali Imran (Associate Editor) earned his Ph. D from the Agriculture University, Faisalabad in 2013 with distinction. His research expertise includes the formulation of the cost-effective polyphenol-based dietary interventions against human maladies. Being a researcher, he has published more than 50 articles in high impact international journals with a cumulative impact factor of more than 80. Further, he has also published 7 book chapters

with international publishers. He is also working as an academic editor in Hindawi groups in the Journal of Biomedical Research International. He has assisted two Ph. Ds and 12 MPhil students within his research domain. He is also working as the section in charge in his HI TEC laboratory.

Contents

**Section 1**

*and Ali Imran*

Malnutrition *by Kathleen Hefferon*

Vitamins

**Section 2**

*by Joseph Kiprop Choge*

*by Francisco Javier Ros Forteza*

*by Junsheng Huo and Chunhong Zhang*

*by Natisha Dukhi*

Malnutrition Risk

Introductory Chapter: Malnutrition

*by Farhan Saeed, Muhammad Imran, Tabussam Tufail*

*by Mohanad Mousa Kareem and Majid S. Jabir*

Biofortification of Crops Using Biotechnology to Alleviate

Malnutrition: Current Challenges and Future Perspectives

**Preface XI**

Introduction to Malnutrition **1**

**Chapter 1 3**

**Chapter 2 9**

**Chapter 3 21**

**Chapter 4 39**

Malnutrition and Global Determinats **65**

**Chapter 5 67**

**Chapter 6 81**

**Chapter 7 97**

Wernicke Encephalopathy in Elderly Related to Severe Malnutrition

Global Prevalence of Malnutrition: Evidence from Literature

Detection of Nutrient-Related SNP to Reveal Individual

## Contents

Preface

This edited volume is a collection of reviewed and relevant research chapters con-

This book covers a very relevant topic that not only proved a major disaster for developing economies but also caused problems in developed economies. Malnutrition causes severe structural and functional abnormalities that hinder the growth of the individual, but also society in general. This book provide complete insight into the

Each contribution comes as a separate chapter complete in itself but directly related

The book is composed of two sections: 1.) Introduction to Malnutrition, and 2.)

The "Introduction to Malnutrition" includes the following chapters: "Introductory Chapter: Malnutrition", "Biofortification of Crops Using Biotechnology to Alleviate Malnutrition", "Vitamins" and "Malnutrition: Current Challenges and Future Perspectives". The second section, "Malnutrition and Global Determinants", includes the following chapters: "Wernicke Encephalopathy in Elderly Related to Severe Malnutrition", "Global Prevalence of Malnutrition: Evidence from Literature" and "Detection of Nutrient-Related SNP to Reveal Individual Malnutrition Risk".

The target audience comprises scholars and specialists in the field. This book will provide deep insights into the problem, pathophysiology and tackling strategies.

> **Muhammad Imran** University of Lahore,

Government College University,

Pakistan

**Ali Imran**

Pakistan

cerning the developments within the malnutrition field of study.

problem, pathophysiology, impact and rectifying strategies.

to the book's topics and objectives.

Malnutrition and Global Determinants.


*2.6. Costs*

The cost was defined as the direct cost related to the surgical procedure (material and medicine costs only). This data was exported from the management accounting REKOLE® analyses that are performed annually at our institution. The standard reimbursement for a TSA was calculated by multiplying the hospital base rate (9550 CHF) by the DRG standard cost-weight, which varied between 1.929 and 2.096 from 2015 to 2019. The standard TSA reimbursement for a patient with basic insurance only was therefore 19,081 CHF in 2015, 20,017 CHF in 2016, 18,651 in 2017, 18,422 CHF in 2018, and 18,479 CHF in 2019. In our consecutive series of patients with basic insurance only (*n* = 47, 38%), the direct cost per case represented 44% of the total cost (44% ± 7%; median, 44%; range, 32–60%). Thus, we considered that 44% of the standard TSA reimbursement should be used as the direct cost reference, which gives: 8396 CHF in 2015, 8807 CHF in 2016, 8206 in 2017, 8106 CHF in 2018, and 8131 CHF in 2019.