**2. Microarrays**

As we described before, the PCR have several advantages over the culture of to identify mi‐ croorganisms from infection. However, the disability to work with different genomes at the same time and the obtaining product with the same molecular size, make the PCR not the better method for diagnosis. Therefore, there have been developed new methods of diagno‐ sis that not only reduce the time process; also they have more sensibility and specificity [14].

That is the case of the DNA microarrays, also called biochip, DNA chip, or gene array, which are defined as an orderly arrangement of samples gene for matching known and un‐ known DNA samples based on base-pairing rules and automating the process of identifying the unknowns and they were created by Brown P.O. y Botstein D. in 1999. An experiment with a single DNA chip can provide to researchers information on thousands of genes si‐ multaneously, a dramatic increase in throughput. Microarray-based technology, with its ad‐ vantage of highly parallel detection, has been applied to both population profiling and to functional studies of complex microbial communities in the environment [15, 16]. In addi‐ tion, several studies have reported the use of PCR-amplified genomic fragment sequences as probes.

In 2005, Yeo *et al.,* reported an outbreak of acute hemorrhagic conjunctivitis in Singapore [11]. Patients were diagnosed clinically with acute hemorrhagic conjunctivitis and it was identified by PCR the presence of an enterovirus and molecular typing confirmed a variant of coxsackievirus A24 (CA24v). Full-length genome sequencing results showed that CA24v virus was responsible for the outbreak and it was evolved from virus emerged 40 years ago.

For identification by PCR/ESI-MS using oligonucleotides specific for bacterial groups rather than to a particular species, although variable regions are amplified between species and strains. Additionally, there are species-specific oligonucleotides used as primers that target genes for antibiotic resistance or some pathogenic characteristics [12]. Subsequent to amplifi‐ cation, amplicons are subjected to mass spectrometry and the pattern obtained is compared with those in the databases. The ability to identify an organism without prior knowledge of the Gram, or group of microorganisms is another advantage [12], since the stains are not re‐ quired or previous isolates that provides fast trial and will always be possible to identify the microorganisms. This technology will be improving the identification of microorganism in ocular infections, it takes some advantages as certainty and specificity, and however the cost

Kaleta *et al.,* designed a study to evaluate the feasibility of the use of PCR/ESI-MS to identify microorganisms directly from blood culture bottles in the clinical microbiology laboratory [12]. The high concordance of the results of this technique with those of standard methods, particularly at the genus level, demonstrates that PCR/ESI-MS technique is capable of rapid‐ ly evaluating clinically complex specimens providing information as to the selection and ad‐

About eye microorganisms, Pedreira *et al.,*evaluated the efficacy of a prophylactic regimen of daily topical 0.5% moxifloxacin and 5% povidone-iodine in patients with Boston type I. The patients with the prophylactic regimen were sampled and analyzed by standard culture methods and by PCR/ESI-MS [13]. The molecular diagnostic approach using PCR/ESI-MS yielded data comparable with those obtained using standard microbiologic techniques. Be‐ cause of the high throughput nature and rapid results, the method might be a useful surveil‐

As we described before, the PCR have several advantages over the culture of to identify mi‐ croorganisms from infection. However, the disability to work with different genomes at the same time and the obtaining product with the same molecular size, make the PCR not the better method for diagnosis. Therefore, there have been developed new methods of diagno‐ sis that not only reduce the time process; also they have more sensibility and specificity [14]. That is the case of the DNA microarrays, also called biochip, DNA chip, or gene array, which are defined as an orderly arrangement of samples gene for matching known and un‐

*1.1.3. PCR coupled to mass spectrometry using electrospray ionization (PCR / ESI-MS)*

is the major disadvantage.

4 Common Eye Infections

ministration of targeted antibiotics.

lance tool in patients with Boston type I.

**2. Microarrays**

The gene arrays can be classified as macroarrays or microarrays, depending on the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contain thousands of spots; microarrays require specialized robotics and imaging equipment.

There are several steps in the design and implementation of a DNA microarray experiment, as is shown in Figure 1.

**Figure 1.** Design and implementation of DNA microarray experiment

It is important to mention that the microarrays made with cDNA are called spotted arrays because the probes are created *in vitro* and the robot put the spots on the microplate. Instead of the microarrays with oligonucleotides which are created *in situ* [17]. Recent studies have used synthesized oligonucleotides as probes because of their flexibility in design and prepa‐ ration; with intensive specificity evaluation applied to the probe design criteria [18].

Both macroarrays and microarrays can have two application forms for the DNA microarray technology: the identification of sequence (gene/gene mutation) and determination of ex‐ pression level of genes.

The determination of expression level of genes the microarrays can study the transcriptome or the proteome. For the transcriptome microarrays the probes consist on cDNA that hybrid‐ ize with the mRNA of the cell. By the other hand, the proteome microarrays can use proteins as probe or the antibody making the antigen-antibody reaction. One example of this micro‐ array is the peptide microarray analysis of *in silico*-predicted epitopes for serological diagno‐ sis of *Toxoplasma gondii* infection in humans [19].

About the identification of gene sequence, microarray should have genomic DNA as probe of a specific chromosome, specifically all the genes that compose the chromosome. Or when a microarray only has a gene with one or more different nucleotides called Single Nucleo‐ tide Polymorphism (SNP) can detect a gene mutation [20].

These microarrays are used to determinate the cancer progression; all the changes on these gene are important to establish a clinical forecast [20]. Such microarrays have been used for the detection of specific bacteria [22, 23], species determination [24], and screening of envi‐ ronmental sequences related to a certain function within a community [25, 26].

Chin-I *et al.,* coupled 16S rDNA PCR and DNA hybridization technology to construct a microar‐ ray for simultaneous detection and discrimination of eight fish pathogens (*Aeromonas hydrophi‐ la, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae* and *Vibrio anguillarum*) commonly encountered in aquaculture. The array comprised short oligonucleotide probes complementary to the poly‐ morphic regions of 16S rRNA genes from the target pathogens. The results showed that each probe consistently identified its corresponding target strain with 100% specificity [27].

Yu-Cheng *et al.,* designed the DNA probes and PCR primers for the detection of *Listeria mon‐ ocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli* O157:H7*, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae*and *Pseudomonas fluorescens* by using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected [28].

An example of microarray designed for infection diagnosis is a microarray developed by Uchida *et al.,* for the direct detection of pathogens in osteoarticular infections by polymerase chain reaction amplification and microarray hybridization [29].

And finally, and the most interesting DNA microarray used for the endophthalmitis diagno‐ sis is the one developed by Tsutomu *et al.* They used 13 samples of vitreous fluid (VF) ob‐ tained from 13 patients during vitrectomy. Vitreous fluids from three patients with suspected endophthalmitis and ten controls without infection were subjected to testing for the presence of bacteria and fungi in culture tests, polymerase chain reaction (PCR) analysis, and DNA microarray analysis. The DNA microarray contained the spots for 16S rDNA, var‐ iable and conserved areas for bacteria, and the 18S rDNA for fungi. No control sample was positive for bacteria or fungi in the culture test, PCR, or microarray analysis. Specimens from two patients (Cases 1 and 2) with suspected endophthalmitis were positive for bacteria in PCR, and a specimen from one patient (Case 3) was positive for fungi in PCR. *Klebsiella pneumonia* (Case 1), *Streptococcus agalactiae* (Case 2), and *Candida parapsilosis* (Case 3) in the PCR-positive specimens were identified by DNA microarray analysis within 24 hours. Cul‐ ture results were also positive for *K. pneumonia* in Case 1, *S. agalactiae* in Case 2, and *C. para‐ psilosis*in Case 3, but required 3 to 4 days to obtain [30].

For infection diagnosis, microarray analysis is complementary to routine cultures for identi‐ fying causative microorganisms and is likely to be a useful tool in patients who require rap‐ id diagnosis and early treatment.


*Proceeding Paper* **Effect of Potassium Formate on Alkali–Silica Reaction in Aggregates with Different Categories of Reactivity †**

**Aneta Antolik \*, Daria Józwiak-Nied ´ ´ zwiedzka , Kinga Dziedzic and Paweł Lisowski**

Institute of Fundamental Technological Research, Polish Academy of Sciences, Adolfa Pawi ´nskiego 5B, 02-106 Warsaw, Poland

**Abstract:** During the wintertime, concrete pavements experience harsh exposure conditions due to the presence of both the freezing–thawing and wetting–drying cycles. Airport concrete pavements are commonly de-iced using chloride-free organic salts such as potassium formate or potassium acetate. However, these materials contain alkali ions which can have harmful effects on both the cement matrix and the aggregate. Specifically, there is an increased risk of occurrence of the alkali–silica reaction (ASR). The goal of this research was to estimate the influence of potassium formate on the potential of causing alkali–silica reaction in aggregates with different categories of reactivity (R0, R1, R2). The accelerated mortar bar test and its modification (which involves replacing sodium hydroxide solution with a potassium formate solution) were used. Detailed SEM-EDS examinations were performed to confirm the presence of alkali–silica reaction and to analyze the influence of potassium formate on the microstructure of mortar.

**Keywords:** alkali–silica reaction; potassium formate; de-icing agent; reactive aggregate

**Citation:** Antolik, A.; Jó ´zwiak-Nied ´zwiedzka, D.; Dziedzic, K.; Lisowski, P. Effect of Potassium Formate on Alkali–Silica Reaction in Aggregates with Different Categories of Reactivity. *Mater. Proc.* **2023**, *13*, 13. https://doi.org/10.3390/materproc<PHONE_NUMBER>

Academic Editors: Katarzyna Mróz, Tomasz Tracz, Tomasz Zdeb and Izabela Hager

Published: 14 February 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/).


*3.2. Effect of ECO in PLA on the Thermomechanical Properties of PLA*

Storage modulus (G') and damping factor (tan δ) were assessed by dynamic mechanical response. In Figure 6, the viscoelastic behavior of PLA\_ECO formulations were exposed. Two characteristic changes in the storage modulus could be distinguished. The first change, between 50 and 70 ◦C, was the drop of storage modulus, which was related to glass transition temperature (Tg) at around 60 ◦C, as reported by Yong et al. [47]. The second change, between 80 and 100 ◦C, was recognized as the beginning of cold crystallization process. The addition of ECO to PLA resulted in a loss of storage modulus at lower temperatures. This was due to the plasticizing effect that ECO exerts on the PLA matrix, which increases the free volume between PLA chains before a saturation effect, decreasing the interaction between them [48]. In addition, at room temperature, neat PLA showed a storage modulus value of 1300 MPa, while the plasticized PLA formulations showed a decrease of this modulus up to 1000 MPa as a consequence of the plasticization effect. On the other hand, the beginning of cold crystallization decreased as ECO content increased, obtaining a shift from 87 up to 84 ◦C for plasticized PLA. This effect was due to plasticizer enabling the rearrangement in packed structure under lower energetic conditions. *Polymers* **2021**, *13*, 1283 10 of 17

**Figure 6.** Plot evolution of dynamic mechanical thermal analysis (DMTA) of PLA with different ECO contents: (**a**) storage modulus (G'); (**b**) damping factor (tan δ). **Figure 6.** Plot evolution of dynamic mechanical thermal analysis (DMTA) of PLA with different ECO contents: (**a**) storage modulus (G'); (**b**) damping factor (tan δ).

The temperature of the tan δ peak was a great manner to obtain an accurate value of Tg which was moved to lower temperatures as ECO content increased. Specifically, the Tg values were reduced from 64.2 (PLA neat) up to 61.9 and 59.5 *°*C for PLA\_5%ECO and PLA\_10%ECO, respectively. Regarding tan delta peak magnitude, it is related to their molecular mobility. As Chieng et al. reported, the addition of plasticizer content led to increase the intensity of tan delta due to higher molecular mobility caused by the plasticizing effect [35]. Then, a significant increase in respect to neat PLA in the magnitude of the damping factor was observed with higher ECO content. On the other hand, Silverajah et al. observed that increasing the content of epoxidized palm oil plasticizer in PLA led to decrease the temperature of tan δ peak [49]. The temperature of the tan δ peak was a great manner to obtain an accurate value of Tg which was moved to lower temperatures as ECO content increased. Specifically, the Tg values were reduced from 64.2 (PLA neat) up to 61.9 and 59.5 ◦C for PLA\_5%ECO and PLA\_10%ECO, respectively. Regarding tan delta peak magnitude, it is related to their molecular mobility. As Chieng et al. reported, the addition of plasticizer content led to increase the intensity of tan delta due to higher molecular mobility caused by the plasticizing effect [35]. Then, a significant increase in respect to neat PLA in the magnitude of the damping factor was observed with higher ECO content. On the other hand, Silverajah et al. observed that increasing the content of epoxidized palm oil plasticizer in PLA led to decrease the temperature of tan δ peak [49].

Table 2 shows a summary of values obtained for Vicat softening temperature (VST) and heat deflection temperature (HDT) of neat PLA and PLA plasticized with ECO. These thermomechanical parameters are directly related to mechanical resistant properties. For this reason, the decreasing trend was the same that had been observed previously in tensile strength and modulus. Regarding VST, a remarkable decrease was detected when ECO content increased. Neat PLA had a VST of 56.6 *°*C, while samples with 7.5 and 10 wt.% ECO, respectively, reduced this value 4.4 *°*C. A similar trend was observed in HDT values, obtaining a difference of 2.6 *°*C between neat PLA and PLA\_10%ECO sample. The Table 2 shows a summary of values obtained for Vicat softening temperature (VST) and heat deflection temperature (HDT) of neat PLA and PLA plasticized with ECO. These thermomechanical parameters are directly related to mechanical resistant properties. For this reason, the decreasing trend was the same that had been observed previously in tensile strength and modulus. Regarding VST, a remarkable decrease was detected when ECO content increased. Neat PLA had a VST of 56.6 ◦C, while samples with 7.5 and 10 wt.%ECO, respectively, reduced this value 4.4 ◦C. A similar trend was observed in HDT values, obtaining a difference of 2.6 ◦C between neat PLA and PLA\_10%ECO sample. The addition

addition of ECO to PLA samples in different percentages facilitated the sliding of the polymeric chains, being also favored by the slight increase in temperature that takes place in

between the PLA and the macromolecules, obtaining the plasticization of the materials

**Table 2.** Vicat softening temperature (VST) and heat deflection temperature (HDT) of PLA with

**Reference VST (°C) HDT (°C)**  PLA 56.6 ± 1.5 52.8 ± 0.5 PLA\_2.5%ECO 55 ± 1.2 51.8 ± 0.6 PLA\_5%ECO 53.2 ± 1.3 50.4 ± 0.4 PLA\_7.5%ECO 52.2 ± 1.1 50.4 ± 0.6 PLA\_10%ECO 52.2 ± 1.4 50.2 ± 0.4

different epoxidized chia seed oil (ECO) content.

[50].

of ECO to PLA samples in different percentages facilitated the sliding of the polymeric chains, being also favored by the slight increase in temperature that takes place in the VST and HDT tests. The ECO molecules decreased the intermolecular attraction forces between the PLA and the macromolecules, obtaining the plasticization of the materials [50].

**Table 2.** Vicat softening temperature (VST) and heat deflection temperature (HDT) of PLA with different epoxidized chia seed oil (ECO) content.


**CORONARY ARTERY DISEASE – NEW INSIGHTS AND NOVEL APPROACHES**

Edited by **Angelo Squeri**

http://dx.doi.org/10.5772/1168 Edited by Angelo Squeri

#### **Contributors**

Wan Zaidah Abdullah, Jun-Jun Wang, Nduna Dzimiri, Ryouta Maeba, Charlotte Lawson, Guoqing Cao, Guillermo Vazquez, Kathryn Smedlund, Jean-Yves Tano, Robert Lee, Mohaddeseh Behjati, Iman Moradi, David Garcia-Dorado, Ignasi Barba, Eva Szaboova, Jian Li, Angela Messmer-Blust, Surjit Singh, Nidhi Gupta

#### **© The Editor(s) and the Author(s) 2012**

The moral rights of the and the author(s) have been asserted.

All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH's written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department (permissions@intechopen.com).

Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html.

#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in Croatia, 2012 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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Cornea and Refractive Concepts

**3**

**Chapter 1**

**Abstract**

visual impairment

**1. Introduction**

Enemy

Infectious Keratitis: The Great

Infectious keratitis tops the list of diseases leading to visual impairment and corneal blindness. Corneal opacities, predominantly caused by infectious keratitis, are the fourth leading cause of blindness globally. In the developed countries, infectious keratitis is usually associated with contact lens wear, but in developing countries, it is commonly caused by trauma during agricultural work. The common causative organisms are bacteria, fungus, Acanthamoeba, and virus. Severe cases can progress rapidly and cause visual impairment or blindness that requires corneal transplantation, evisceration, or enucleation. The precise clinical diagnosis, accurate diagnostic tools, and timely appropriate management are important to reduce the morbidity associated with infectious keratitis. Despite the advancement of diagnostic tools and antimicrobial drugs, outcomes remain poor secondary to corneal melting, scarring, or perforation. Eye care strategies should focus on corneal ulcer prevention. This review addresses the epidemiology, diagnostic approach, clinical manifestations, risk factors, investigations, treatments, and the update of major

*Vatookarn Roongpoovapatr, Pinnita Prabhasawat,*

*Saichin Isipradit, Mohamed Abou Shousha*

clinical trials about common pathogens of infectious keratitis.

**Keywords:** infectious keratitis, corneal ulcer, corneal scar, blindness,

Infectious keratitis tops the list of diseases leading to visual impairment and corneal blindness. Globally, it is approximated that 1.3 billion people live with visual impairment [1]. The major causes of corneal blindness included infectious keratitis, ocular trauma, trachoma, bullous keratopathy, corneal degenerations, and vitamin A deficiency [2]. Corneal opacities, predominantly caused by infectious keratitis, are the fourth leading cause of blindness globally [3]. Recent paper reported that 3.5% of global blindness could be attributed to corneal opacity [4]. According to the goal of the "Vision 2020: The Right to Sight," which was proposed by WHO, the prevention of avoidable corneal blindness should receive more awareness [5, 6]. Interestingly, the majority of visual impairment is avoidable [1]. Infectious keratitis accounts for 10% of avoidable visual impairment in the world's least-developed countries [3]. The number of people with avoidable visual loss has increased considerably because of population growth and aging. This trend will continue beyond

*and Puwat Charukamnoetkanok*


**4. Results**

The results below include a description of the artworks produced, and the results of the evaluation of the works by the critique groups with a focus on the last critique. Photographs of the animated three- and four-dimensional images are shown below for illustrative purposes only as it is very di fficult to visualize these artworks in two dimensions. Figure 1a,b are of the monochromatic analogue shadowgrams. Figure 1a *Great Great Grandfather* depicts a holographic image of a man, contained within an antique compass, striding along with a walking stick. The image looks to have been taken in the 1940s. The transparent holographic film on which the image is produced is placed on top of the glass protecting the compass needle which is clearly visible beneath it. The image of the walker appears to be sunk beneath the glass existing in the same space as the compass needle. Figure 2b depicts two portraits of the same woman 50 years apart. A transparent hologram of a photographic portrait taken in the 1940s sits on top of a digital photographic portrait. The earlier holographic portrait is sunk beneath the surface of the later photograph.

**Figure 1.** (**a**) *Great Great Grandfather* 2014, mixed media with reflection hologram, 8 cm × 20 cm, P. John and (**b**) *Great Grandmother*, 2014, mixed media with reflection hologram, 12.7 cm × 15.2 cm, P. John created and photographed by the author.

The first two holograms in Figure 1 *Great Great Grandfather* and *Great Grandmother* were shown to both the small group of contemporary artists working in film and media in 2014 and to the group of experts in art and holography at ISDH2018. To see the holograms, the viewers had to move the artworks around in order to illuminate the embedded holograms at the correct viewing angle. The comments from the artists focused more on the emotional impact of the work and of the medium, rather than the content of the holograms. The artists were unable to determine differences in the depth of holographic space used in the works. As a result, the author chose an audience of experts in art and holography to evaluate the last artwork produced in the study.

*Passing Time, Distant Memory* shown below in Figure 2a,b was created during the very last phase of the study and critiqued by the art and holography experts only. The digital animated hologram was hung and lit as part of the exhibition in City Art Gallery, Aveiro, Portugal. As the viewer moves laterally in front of the hologram from left to right, the photographs swing in an arc to show spaces and distances between them; the images on the left become obscured by those on the right-hand-side as if the photographs were solid. The images farthest from the viewer on the left-hand-side of the image depicted older photographs and these were sunk deeper within the *Z*-axis of holographic space. The image, an animated video on the far right-hand side was the most recent. Seven seconds of footage taken from a wedding video were edited to produce an animation in which the bride is shown to speak and laugh as the viewer moves. The video sits on a virtual surface which projects in front of the image plane of the hologram, protruding into the viewer's space.

(**b**)

**Figure 2.** (**a**) First view of *Passing Time, a Distant Memory*, 2018, digital animated hologram, 65 cm × 25 cm, P. John. (**b**) Second view of *Passing Time, a Distant Memory*, 2018, digital animated hologram, 65 cm × 25 cm. Produced and photographed by the author.

The purpose of the final silent researcher critique was to determine whether the audience could perceive and comprehend the concept that the *Z*-axis of holographic space depicted a chronological narrative; whether the viewers had a new experience in viewing the work; and whether it had an affective impact. The results demonstrated the following: that the experts thought that the artwork was novel; depicted images and memories within holographic space in a new manner; and had affective impact. They described their experience of the work was as follows:

"A new way to present the past." (Expert I); "You are sinking memories into holographic space." (Expert G); "I felt sad when I looked at the image." (Expert A).

The audience of experts recognised that they were part of the time-line and that the *Z*-axis of holographic space included their own present time and space:

" ... if we're thinking we're dealing with a timeline are we were part of that line by viewing the work?" [Laughter] (Expert B); "Okay. I mean it's about this moment. It's about this very particular moment." (Expert D).

The experts discussed different concepts of time inherent in the work they were shown and included their felt experience of time and the concept of linear time.

One of the study's aims was that the work should have an affective impact. During the silent researcher critique, participants reported feeling sadness and nostalgia. However, as the critique

continued enabling people to talk without discussion, other emotional responses to the works became evident: there was slight discomfort, and then when the group broke the 'no discussion' guidance there was disagreement. Two experts expressed that they distrusted that the family history included in the artwork was authentic. A sense of tension during the silent researcher critique was relieved with laughter when there was a suggestion by one expert that the family in the artwork was not actually the author's and that the narrative presented as authentic was fictional. Another area of discomfort was due to three of the art and holography experts disagreeing with one another regarding issues of metaphysics and physics. Two participants shared concepts of objects containing memories, or the essence of the person that they related to. One art expert jokingly said that the objects contained ghosts, and another, whose training had been rooted in science, firmly disagreed. Two contemporary artists in the pilot 'Questions' session evaluation had discussed the same concepts of objects containing memories; however, the research artefacts did not contain the author's memories; most of the photographs included in the artworks were taken before the author was born. Instead, the photographs suggest memories, someone's memories.


**Celebrating 120 Years of Butantan Institute Contributions for Toxinology**

Editor

**Ana M. Moura-Da-Silva**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Ana M. Moura-Da-Silva Instituto Butantan Brazil

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Toxins* (ISSN 2072-6651) (available at: http://www.mdpi.com).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-3188-5 (Hbk) ISBN 978-3-0365-3189-2 (PDF)**

Cover image courtesy of Butantan Institute.

© 2022 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.


*3.2. Fullerene Onion-Anions*

We now move to the discussion of the wavefunctions of the valence electron in the fullerene onion-anions (C60@C240)− , (C60@C540)−, (C240@C540)− and (C60@C240@C540)−. We note first that the potentials of these fullerene onions are, obviously, either double-well or triple-well potentials. Correspondingly, one can expect a greater number of bound states available to the attached electron in these fullerene onion-anions. In our case, the calculations predicted the existence of only two discrete *p*-states—the 2*p* and 3*p* excited states—, in contrast to only the 2*p* excited-state in the bare fullerene anions. The corresponding *P*1*s*, *P*2*p* and *P*3*p* functions are plotted in Figure 3 where a number of new features are exhibited.

The most striking discovery relates to the behavior of the *P*3*p* excited-state wave functions. Their highly peculiar behavior is completely different from the behavior of the *P*1*s* and *P*2*p* functions in any of these fullerene onion-anions. Indeed, we find that a significant part of the *P*3*p* function and, thus, the electron density of the attached electron, is packed inside the wall of the inner cage directly adjacent to a larger fullerene cage in each of these double- and triple-cage fullerene onion-anions. Although this is evident from Figure 3, this is also supported by looking at the mean radii, *r*¯3*p*, of the 3*p* orbitals in these fullerene onion-anions as well. The calculated *r*¯3*p*'s are: *r*¯3*p* ≈ 7 in both (C60@C540)−

and (C60@C240)− (thus, the 3*p* orbital falls into the C60 potential well), whereas *r*¯3*p* ≈ 14 in both (C240@C540)− and (C60@C240@C540)− (thus, *r*¯3*p* falls into the C240 potential well). This is in a sharp contrast to the *P*1*s* and *P*2*p* functions that are mainly packed in the potential well of the largest fullerene cage (C540, in our case) in corresponding fullerene onion-anions, respectively, as is evident from Figure 3 (also, *r*¯1*s* ≈ *r*¯2*p* ≈ 20 in all fullerene onion-anions under discussion). Especially surprising is the behavior of the *P*3*p* function in (C60@C540)−, where its probability density is almost entirely located inside C60, despite the size of C60 being significantly smaller than C540, so that the C60 potential well should not have affected the attached valence electron at all, as in the case of the *P*1*s* and *P*2*p* functions (see Figure 3c,d). In any case, the behavior of *P*3*p* in these fullerene onion-anions is extraordinary, a complete break with conventional wisdom.

**Figure 3.** Calculated radial ground-state *P*1*s*(*r*) and excited-state *P*2*p* and *P*3*p* (due to the 1*s* → *np* transitions, *n* = 2, 3) of the attached electron in fullerene onion-anions: (**a**) (C60@C240)−, (**b**) (C240@C540)−, (**c**) (C60@C540)−, (**d**) (C60@C240@C540)−: solid, *P*2*p*; dashed, *P*1*s*; dash–dot, *P*3*p*. Also plotted are the *P*1*s* (dash–dot–dot) functions in bare C−240 and C−540, as designated, for comparison purposes. Note, to avoid any confusion, that the graphs for the *P*1*s* and *P*2*p* functions in the fullerene onion-anions tightly overlap with each other and are practically indistinguishable from each other with some exception in the case of (C60@C240)−.

We interpret the predicted behavior of the *P*3*p* excited-state functions in the fullerene onion-anions as being due to both the multi-well nature of the fullerene onion-anion potentials and the fact that, in contrast to the nodeless *P*1*s* and *P*2*p* functions, the *P*3*p* function has one node. That is, the *P*3*p* function is distinctly split into an inner part (before the node) and an outer part (beyond the node). It appears that the inner part of the *P*3*p* function falls into the potential well associated with a fullerene cage adjacent to the outermost cage in the fullerene onion-anion. Thus, the attached electron partially resides in the inner well.

We note, though, that the behavior of the *P*3*p* function in the fullerene onion-anions is somewhat reminiscent of the behavior of the excited *P*3*d* and *P*4*d* functions, excited from the 3*p* subshell, in endohedral calcium, Ca@C60 [30]. There, a significant transfer of the 4*d*, but not 3*d*, electron density into the inner space of C60 was demonstrated. That resulted in a significant increase in the amplitude of the *P*4*d* orbital in the inner space of C60. Consequently, the mean radius of the 4*d* orbital was reduced from *r*¯4*d* ≈ 14 in free Ca to only *r*¯4*d* ≈ 4.3 < *rc* = 5.8 in Ca@C60 [30]. That situation, in turn, was commented on to be somewhat reminiscent of the behavior of the excited 4 *f* and 5 *f* orbitals in Ba+ [31,32] that was shown to be due to the double-well nature of the potential of Ba+ that caused partial orbital collapse of 5 *f* into the inner well, thereby causing 5 *f* , rather than 4 *f* , to have the greater amplitude near *r* = 0.

#### *3.3. Oscillator Strengths and Photodetachment Cross Sections*

Calculated oscillator strengths, *f*1*s*→*np*, of the C−60, C−240, and C−540 bare fullerene anions as well as the (C60@C240)−, (C60@C540)−, (C240 @ C540)−, and (C60@C240@C540)− fullerene onion-anions are/were listed in Table 1 which contains a wealth of information. Since a principal goal of this work is to explore the spectral distribution of oscillator strength, we focus on a comparison among the total oscillator strengths of the discrete spectra, i.e., *f*1*s*→2*p* + *f*1*s*→3*p* ≡ *f*(2*p*+3*p*), for the single and nested fullerene cages Thus, as we transition from C−240 → (C60@C240)−, the *f*(2*p*+3*p*) oscillator strength is increased. The same change in *f*(2*p*+3*p*) is characteristic along all other transition paths as well: C−540 → (C240@C540)−, C−540 → (C60@C240@C540)−, and, in principle, C−540 → (C60@C540)−, too. Hence, we have unraveled a general tendency: stuffing of a bigger fullerene cage with a smaller fullerene cage, as well as progressively stuffing the biggest fullerene cage with several smaller fullerene cages, results in the transfer of a part of the oscillator strength from a continuum spectrum into a discrete spectrum.

Now, how does the discovered tendency affect the 1*s*-photodetachment cross section, *σ*1*s*? Obviously, the total area under the graph for *σ*1*s* should be decreasing along the discussed fullerene transition paths. This may result in the disappearance of some of the resonance structures in *σ*1*s*, or making them narrower, or decreasing their heights, or lowering the values of other parts of *σ*1*s*, or all of the above cumulatively. It is, therefore, extremely interesting to study the modifications in *σ*1*s*'s on a comparative one-to-one basis for different fullerene anions.

Calculated *σ*1*s*'s for C−240 versus (C60@C240)−, as well as C−540 versus (C60@C540)−, (C240@C540)−, and (C60@C240@C540)− are depicted in Figure 4 as functions of the photoelectron momentum *κ*, in order to eliminate the impact of differences in 1*s* ionization potentials between the fullerene anions on details of *σ*1*s*'s, for the adequacy of the comparison between these anions.

We first note that the calculated cross sections exhibit the oscillatory behavior versus the photoelectron momentum, *k*. Such resonances have been well understood for both photoionization and photodetachment of, as well as electron scattering by, fullerene and endo-fullerene complexes in a large body of research; we refer the reader to the above references, to the review paper [8] for many more references on the subject, as well as, e.g., to [4–8,10–24,29,31,38,39,46] from [21] (and references therein). Following [33], these resonances are commonly referred to as the *confinement resonances*.

**Figure 4.** Calculated *σ*1*s* photodetachment cross sections of bare fullerene anions and nested fullerene onion-anions, as designated in the figure. Note, on all parts of the figure, the *σ*1*s* of C−60 is represented by a dashed-line.

Secondly, note that the prediction mentioned above on the modification of the photodetachment cross section along the path from the bare fullerene anions to the doubleand triple-cage fullerene onion-anions is seen to be correct. Indeed, we see the disappearance of one or even two confinement resonance structures (near the lower energy end of the spectrum), and the significant decrease in their amplitudes (except for the case of the (C60@C540)− onion-anion, which is an extraordinary case anyway, as was discussed above). Furthermore, it is interesting that the resonances in *σ*1*s* of (C60@C540)− are seen to be shifted towards higher *k*'s, compared to *σ*1*s* of the bare (C540)− anion, whereas in all other nested fullerene onion-anions they shift toward lower *k*'s, compared to corresponding bare counterparts.

Thirdly, it is quite interesting that *σ*1*s*'s of all fullerene onion-anions, whether doublecage or triple-cage anions, do not differ much in magnitude from *σ*1*s* of the smallest C−60 anion in this sequence of fullerene anions (except for *σ*1*s* of extraordinary (C60@C540)− at a lower end of the spectrum). To emphasize this, we added *σ*1*s* of C−60 to all plots in Figure 4 to facilitate this comparison. One can see that *σ*1*s*'s, associated with the nested

fullerene onion-anions, oscillate around *σ*1*s* of C−60 with average amplitudes that are not much different from *σ*1*s* of C−60.

Lastly, we note that, to check the connection of calculated *σ*1*s*'s to calculated oscillator strengths, we calculated the oscillator strength of the continuum spectrum, *f*1*s*→ *p*, by appropriately integrating *σ*1*s*'s in accordance with Equation (7). These calculated *f*1*s*→ *p*'s are presented in Table 1, and the fact that they have the same values as those obtained from the oscillator sum rule ( *f*1*s*→ *p* = 1 − *f*1*s*→(2*p*+3*p*)) speaks to the accuracy of the calculated *σ*1*s*'s.