**About the Authors**

#### **Camlin, David**

Dave Camlin's musical practice spans performance, composition, teaching, socially engaged music practice, and research. A singer/songwriter by trade, his musical practice is motivated by the idea of music making as a creative resource for fostering kinship and living more sustainably, as a potent form of civic imagination. He lectures in music education at the Royal College of Music and Trinity-Laban Conservatoire and was Head of Higher Education and Research at Sage Gateshead from 2010 to 2019. His research explores the relationship between aesthetic and participatory traditions of music making and highlights how the implicit tensions within these contrasting perspectives can be resolved through a foregrounding of their "paramusical" benefits and effects. His research focuses on group singing, music health and wellbeing, musician education, music and virtuality, and Community Music (CM). He has pioneered the use of Sensemaker® "distributed ethnography" as a method for research into cultural phenomena and has won the National Trust's Outstanding Achievement Award in 2019 for an AHRC / Arts Council England–funded mountaintop singing project, The Fellowship of Hill and Wind and Sunshine, and was co-chair of the Special Interest Group (SIG) in singing and mental health for the March Network.

#### **Davidson, Jane W.**

Jane W. Davidson is Professor of Creative and Performing Arts at the Faculty of Fine Arts and Music and Chair of the Creativity and Wellbeing Hallmark Initiative, University of Melbourne. Her scholarship has contributed to the study of musical skill, music as social interaction, arts for wellbeing outcomes, music and emotion, and opera performance. She has published extensively and has been a recipient of research grants in Australia and internationally. She is also an opera director, currently coordinating the MMus in Opera Performance. Previous roles at the University of Melbourne include Acting Director of the Victorian College of the Arts, Head of Performing Arts, Associate Dean Research, and Associate Dean Engagement. She has also been Deputy Director of the Australian Research Council's Centre of Excellence for the History of Emotions, President of the Australian Music & Psychology Society, Vice-President of the European Society for the Cognitive Sciences of Music and Editor of the academic journal Psychology of Music. She is a Fellow of the Australian Academy of the Humanities.

#### **Haddon, Elizabeth**

Elizabeth Haddon SFHEA, LRSM, is a Senior Lecturer at the University of York, where she devised and leads the MA in Music Education: Instrumental and Vocal Teaching. Her research focuses on instrumental pedagogy, creativity, and musical performance, particularly in higher music education, and her output includes *Making Music in Britain: Interviews with those behind the notes* (Ashgate 2006), as well as two co-edited books with Pamela Burnard: Activating Diverse Musical Creativities: *Teaching and Learning in Higher Music Education* (Bloomsbury, 2015) and *Creative Teaching for Creative Learning in Higher Music Education* (Routledge, 2016). Her published research also includes the exploration of empathy and partnership in piano duet playing and performance with Mark Hutchinson, mental health in the higher music education context, and investigating pedagogical approaches across cultures.

#### **Heyde, Neil**

Neil Heyde is the cellist of the Kreutzer Quartet and Professor of Music and Head of Postgraduate Programmes at the Royal Academy of Music. As a soloist and chamber musician, he has appeared throughout Europe and in China, the USA, and Australia, broadcasting on the major radio networks and recording more than 40 discs, many in collaboration with composers. This experience is central to his research, which focuses on exploring the ways in which musicians communicate with one another and relationships between performers and composers, past and present. He has published on collaborative relationships, instrumental choreography, and issues in interpretation. He is editing Debussy's three late sonatas for the *Œuvres completes de Claude Debussy* and is working on a series of interconnected projects that explore some of the ways in which instruments are "not just tools". He is completing a solo project with composer Richard Beaudoin on a series of six pieces, each a form of transcription of an iconic recording: Argerich playing Chopin, Casals playing Bach, Debussy playing Debussy, Gould playing Schoenberg, Monk improvising on Johnny Green, and Maggie Teyte and Alfred Cortot performing Debussy. His teaching draws extensively on recordings as a means for discovering the richness of musical relationships.

#### **Hunter, Mary**

Mary Hunter is A. Leroy Greason Professor of Music Emerita at Bowdoin College in Maine, USA, where she taught music history, music theory, and courses on Middle Eastern music, music and gender, and music in everyday life. She has written books and articles on 18th-century opera, 18th- and early 19th-century chamber music, and on the ways the ideologies of classical music play into the experiences and language of performers. She is an active amateur violinist.

#### **Hutchinson, Mark**

Mark Hutchinson is a Lecturer in Music at the University of York, where he is a member of the Contemporary Music Research Centre and the Musical Cultures and Communities Research Cluster. His research focuses upon creative approaches towards the analysis of recent contemporary music. His book Coherence in New Music: Experience, Aesthetics, Analysis (Ashgate, 2016) uses ideas from a variety of different disciplines to argue for a novel concept of coherence within recent classical music. He has published articles examining overlaps between music, literature, and philosophy in works by Henri Dutilleux, Toru Takemitsu, and Georg Friedrich Haas. He is also active as a piano ¯ accompanist, chamber musician, and oboist.

#### **Kanga, Zubin**

Zubin Kanga is a pianist, composer, researcher, lecturer, and technologist. His work in recent years has focused on new interactions between live musicians and new technologies, including motion and bio-sensors, AI, analogue synthesizers, new interactive instruments, magnetic resonators, interactions with live-video, and internet-based scores. Zubin has premiered 120 works and performed at many international festivals, including the BBC Proms, London Contemporary Music Festival, Huddersfield Contemporary Music Festival (UK) Melbourne Festival (Australia), Festival Presences (France), Klang Festival (Denmark), Podium Festival (Germany), Resonator ´ Festival (Sweden), November Music (Netherlands), and Borealis Festival (Norway). Recent collaborations include those with Alexander Schubert on his internet-based work *WIKI-PIANO.NET*, with Nicole Lizee on her ´ *Scorsese Etudes*, and with Philip Venables on *Answer Machine Tape*, 1987, a concert-length piano and multimedia work exploring the life of artist David Wojnarowicz. After graduating from the University of Sydney and the Royal Academy of Music, Zubin was a postdoctoral researcher at the University of Nice and IRCAM and a Leverhulme Early Career Fellow at Royal Holloway, University of London, where he is now a Lecturer in Musical Performance and Digital Arts. He is also the Principal Investigator of Cyborg Soloists, a UKRI Future Leaders Fellowship–funded project exploring new music–technology collaborations between artists and industry.

#### **King, Elaine**

Elaine King is a Reader in Music at the University of Hull. She is a performer and musicologist with research interests in performance studies, music psychology, music education, and music analysis. Her doctoral thesis focused on the nature and effects of interaction in cello–piano duos during ensemble rehearsal and performance. Since then, she has published widely on different aspects of solo and ensemble playing, including breathing, rehearsal strategies, gestures, and small group work. She has co-edited four books with Ashgate/Routledge Press: *Music and Gesture* (2006), *New Perspectives on Music and Gesture* (2011), *Music and Familiarity* (2013), and *Music and Empathy* (2017). She is currently working on three projects: STROKESTRA (in partnership with the Royal Philharmonic Orchestra), Chinese Whispers (language learning through choral singing), and Transitions in Music Education. She is the Associate Editor of the journal *Psychology of Music*, Action Editor for *Music and Science* and a member of the Editorial Board for the *Journal of the Royal Musical Association*. As a performer, she is a cellist, pianist, and conductor. She directs the University Camerata and is involved in other regional chamber ensembles and orchestras when time allows.

#### **Kjar, David**

Performance studies scholar David Kjar is an Associate Professor of Core Studies and Music History in the Chicago College of Performing Arts at Roosevelt University. He has performed with early music ensembles in Europe and South and North America. He holds a Master's degree in historical performance (Natural Trumpet) from the Royal Conservatory of The Hague and a PhD in musicology from Boston University. His research grounds theories and philosophies on early music in specific performance experiences (performers and listeners), reframing the early music movement as a sonically constructed and heard Other Performance. He presents internationally on the topic and has published on Wanda Landowska, the early music movement, and authenticity in 21st-century contexts. He is committed to better understanding what constitutes a 21st-century artist, especially within conservatory training. The Paul R. Judy Center for Innovation and Research from the Eastman Institute for Music Leadership (Eastman School of Music) recognized this commitment with an innovation grant. He is the Executive Director of the Center for Arts Leadership at the Chicago College of Performing Arts, helping emerging professionals cultivate artistically and financially sustainable career paths. He is also a member of the international editorial board of the forthcoming journal *Performing Arts Histories*.

#### **Krause, Amanda E.**

Amanda E. Krause is Lecturer in Psychology at the College of Healthcare Sciences, James Cook University, Queensland. As a music psychology scholar, she studies how we experience music in our everyday lives from a psychological perspective. Findings from her research have made significant contributions to understanding how listening technologies influence people and how our musical experiences impact health and well-being. She is the author of numerous academic publications and currently serves as the President of the Australian Music & Psychology Society. She conducts community-focused and community-engaged research, working with music industry companies, performing arts organisations, mental health practices, radio providers, and aged-care facilities.

#### **Krivenski, Maria**

Maria Krivenski is a conservatoire-trained classical pianist, with a wide performing experience both as a soloist and chamber musician. She is also a music lecturer and piano tutor at Goldsmiths, University of London, where she has received the Peake Award for Excellence in Learning and Teaching. As a researcher, Maria has focused on the teaching/learning practices and the culture of musical performance in a university context. Her study, "'Feeding back' in musical performance: exploring feedback practice in relation to students' and tutors' learning and teaching experience", was funded by the PALATINE Development Award scheme. She has participated in the large-scale, EU-funded research projected PRAISE, contributing to the development of a social timeline for musical performers that facilitates online tutor and peer feedback. Maria has been awarded a PhD from the University of Sheffield for her research on emic understandings of classical musical performance in a university context.

#### **McCaleb, Murphy**

Murphy McCaleb is a senior lecturer in Music at York St. John University. He received his doctorate in ensemble performance studies from the Royal Birmingham Conservatoire after studying trombone performance and chamber music at the University of Alaska and the University of Michigan. As a bass trombonist and pianist, Murphy engages in a wide range of music, including classical, jazz, rock, folk, electronic, and experimental and has performed in two Glastonbury Festivals as part of the New York Brass Band. He has recorded on multiple albums, most recently *Mythical and Angry*, a funk collaboration with progressive rock drummer Andy Edwards, and Flora Greysteel's *From the Ground*. His background in musical direction includes an installation at the Illuminating York festival in 2016 and a four-star-reviewed run of Gavin Bryars' Jesus' Blood Never Failed Me Yet at the Edinburgh Fringe in 2019. His first book, *Embodied Knowledge in Ensemble Performance*, was released by Ashgate in March 2014.

#### **Montanari, Allegra**

Allegra Montanari enjoys a multi-faceted career as a change management consultant, nonprofit entrepreneur, creative leader, and professional cellist. She currently supports the Business Advisory Service practice at Slalom, a global consulting firm focused on strategy, business transformation, and technology. Before finding her way to consulting, Ms. Montanari founded the nonprofit Sharing Notes, which has since delivered live music to over 35,000 people hospitalized throughout Chicago. She served as Executive Director for eight years, growing the organisation from a small student group to an award-winning nonprofit. Appointed to the leadership of Roosevelt University's Chicago College of Performing Arts in 2017, Allegra combined her background of teaching, entrepreneurship, and performance to serve as the founding Director of the Center for Arts Leadership. Allegra has led presentations of the Civic Orchestra of Chicago, Indiana University's Jacobs School of Music, the College Music Society's National Conference, and the Royal Music Association (UK). As a professional cellist, Ms. Montanari has performed with ensembles and artists across the country. Ms. Montanari holds degrees in music and business from Indiana University and completed her Master's degree at the Chicago College of Performing Arts, where she was a student of John Sharp, Principal Cellist of the Chicago Symphony.

#### **Todd, Rae W.**

Rae W. Todd is a freelance clarinettist and independent researcher. He completed his PhD in Music Performance at the University of Hull in 2020 with a thesis on the phenomenon of play in professional chamber ensemble rehearsals and a portfolio of performances that featured a recital with the Berkeley Ensemble. He has performed widely across the UK as a soloist and chamber musician, including in the Edinburgh Fringe Festival, the Lincoln International Chamber Music Festival, and the Red Cross International Summer Concert Series. As an orchestral musician, he has performed with the Welsh National Orchestra, the Yorkshire Symphony Orchestra, Culture: Orchestra, Hull University Camerata, the York Guildhall Orchestra, and the Royal Welsh College of Music and Drama Orchestra and Wind Ensemble. He continues to pursue research on aspects of music making when time allows.

#### **Thomas, Kerry**

Kerry Thomas received her Bachelor of Musical Arts in the spring of 2022 in Music and English from Roosevelt University. Kerry has worked for the Chicago College of Performing Arts Center for Arts Leadership since 2018, first as a research assistant and currently as its Grants Manager. A trumpet player, Kerry has interests in both musicological research and performance. She recently completed her Honors undergraduate thesis, "Composing the Grand Ole Opera: Hidden Critiques of Southern Life, Liturgy, and Literature in the Operas of Carlisle Floyd." In the thesis, Kerry reveals how Floyd glosses the source material for his operas to explicitly comment on the United States, particularly the American South, arguing that Floyd's operas can be taken to be overtly political and, at times, as forming explicit critiques on American life. Kerry will continue her musicological pursuits in graduate school.

#### **Waddington-Jones, Caroline**

Caroline Waddington-Jones is a Lecturer in Music Education at the University of York. She has published in various leading peer-reviewed journals in music education and psychology, as well as guest-editing a Special Issue of Empirical Musicology Review and co-editing a volume on music and empathy research for Routledge with Dr. Elaine King. Caroline's research interests include technology and music learning; music and wellbeing; equity, diversity, and inclusion in music education; and music and empathy. She is currently the PI of two research projects. The SMILE research project (funded by Paul Hamlyn Foundation and SEMPRE) aims to develop and evaluate a new online resource for staff in special schools to help to address the current equality gap in music provision for disabled pupils. Meanwhile, the CoMusicate project (funded by Closing the Gap Network) works with people with lived experience of severe mental illness to design new technology that will support social and creative interaction through music and sound. Caroline has a strong commitment to musical inclusion and works with organisations such as Live Music Now, Music in Hospitals and Care, The Amber Trust, and Sounds of Intent to bring more music learning opportunities to disabled children and young people.


*Article* **Fabrication of Ultrathin MoS2 Nanosheets and Application on Adsorption of Organic Pollutants and Heavy Metals**

**Siyi Huang 1, Ziyun You 1, Yanting Jiang 1, Fuxiang Zhang 1, Kaiyang Liu 1, Yifan Liu 1, Xiaochen Chen 1 and Yuancai Lv 1,2,\***

Received: 10 April 2020; Accepted: 24 April 2020; Published: 26 April 2020

**Abstract:** Owing to their peculiar structural characteristics and potential applications in various fields, the ultrathin MoS2 nanosheets, a typical two-dimensional material, have attracted numerous attentions. In this paper, a hybrid strategy with combination of quenching process and liquid-based exfoliation was employed to fabricate the ultrathin MoS2 nanosheets (MoS2 NS). The obtained MoS2 NS still maintained hexagonal phase (2H-MoS2) and exhibited evident thin layer-structure (1–2 layers) with inconspicuous wrinkle. Besides, the MoS2 NS dispersion showed excellent stability (over 60 days) and high concentration (0.65 ± 0.04 mg mL−1). The MoS2 NS dispersion also displayed evident optical properties, with two characteristic peaks at 615 and 670 nm, and could be quantitatively analyzed with the absorbance at 615 nm in the range of 0.01–0.5 mg mL−1. The adsorption experiments showed that the as-prepared MoS2 NS also exhibited remarkable adsorption performance on the dyes (344.8 and 123.5 mg g−1 of *qm* for methylene blue and methyl orange, respectively) and heavy metals (185.2, 169.5, and 70.4 mg g−1 of *qm* for Cd2+, Cu2+, and Ag+). During the adsorption, the main adsorption mechanisms involved the synergism of physical hole-filling effects and electrostatic interactions. This work provided an effective way for the large-scale fabrication of the two-dimensional nanosheets of transition metal dichalcogenides (TMDs) by liquid exfoliation.

**Keywords:** transition metal dichalcogenides; liquid exfoliation; adsorption; quenching

### **1. Introduction**

Given the special structure and potential applications, two-dimensional materials have drawn plenty of concerns [1,2], such as graphene, boron nitride, and molybdenum disulfide. Among them, the ultrathin molybdenum disulfide (MoS2) nanosheets, which exhibit an evident layered structure, have attracted ample attentions because of their excellent performance on several fields, such as catalysis, sensors, and pollution remediation [1,3]. Recently, the ultrathin MoS2 nanosheets were reported to show excellent prospects in pollution control [3,4]. Therefore, it was urgent to explore an effective method to produce ultrathin MoS2 nanosheets.

To date, a few methods have been reported for efficient preparation of ultrathin MoS2 nanosheets [5–10], for example, mechanical exfoliation, sputtering, atomic layer deposition, chemical methods, and liquid-based exfoliation. In spite of the excellent performance of the prepared monolayer or few-layer MoS2 nanosheets through mechanical methods, the production efficiency was rather low, which severely limited the large-scale applications. Meanwhile, although most of the chemical methods like hydrothermal and solvent thermal routes could produce large-scale few-layer MoS2 nanosheets, they generally needed strict reaction control, such as high temperature and pressure. Instead, due to the controllable operation and high production, liquid-based exfoliation was regarded as the most promising way for the production of ultrathin MoS2 in large scale. According to previous studies [7,8], the solvent showed a significant impact on the exfoliation of MoS2. Among which, pyrrolidone-based solvents like *N*-methyl-2-pyrrolidone displayed excellent MoS2 exfoliation efficiency with 0.3 mg mL−1 of MoS2 nanosheets concentration, because they showed similar surface energy with MoS2. Nevertheless, considering their significant environmental risk, high toxicity and high-boiling points of the pyrrolidone-based solvents probably limited the large-scale application. To replace these toxic solvents, a number of polar solvents that own low boiling point and molecular weight were tested, such as water, methanol, ethanol, and isopropanol [11,12]. Unfortunately, given the different surface energy between the MoS2 and polar solvents, most of the polar solvents showed dissatisfactory exfoliation efficiency [11]. Interestingly, it was reported that the MoS2 exfoliation efficiency in the mixed solution with two of the polar micromolecular solvents was much better than those in the single solvent [13,14]. Meanwhile, it was worth noting that the MoS2 exfoliation efficiency could be significantly improved when some organic small molecules, surfactants, or polymers were added in the polar micromolecular solvents, such as sodium cholate, Tween 80, Tween 85, sodium naphthalenide, Brij 30, Brij 700, Triton X-100, and so on [15–17]. Nevertheless, the strong van der Waals interaction between the MoS2 layers still limited the MoS2 nanosheets production.

Recently, owing to the effective break of the van derWaals force between the MoS2 layers, quenching was found to be an effective way to exfoliate the graphene analogues [18–21]. Previous investigation showed that the high-quality ultrathin graphene sheets were fabricated by rapidly cooling the hot bulk graphite and pre-expanded graphite in aqueous solutions of NH4HCO3 and hydrazine hydrate, respectively [19,20]. Meanwhile, the boron nitride and MoS2 nanosheets were also synthesized via the rapid quenching of hot bulk boron nitride and MoS2 in the liquid N2 [18,21]. Although the production efficiency of the nanosheets was dissatisfactory, we suspect that if the bulk MoS2 was pretreated with the quenching process, the exfoliation efficiency of MoS2 nanosheets in the polar micromolecular solvents could be significantly improved.

Herein, a hybrid strategy with the combination of quenching process and liquid-based exfoliation was employed to fabricate the ultrathin MoS2 nanosheets. The microstructures, morphology, and optical properties were analyzed. In addition, the adsorption performance of dyes and heavy metals was also discussed.

### **2. Material and Methods**

### *2.1. Materials*

Ammonium tetrathiomolybdate ((NH4)2MoS4) was provided by Sam Chemical Technology Co., Ltd. (Shanghai, China). Hydrazine monohydrate (N2H4·H2O) was provided by Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium hydroxide (NaOH), sulfuric acid (H2SO4), nitric acid (HNO3), methylene blue (MB), methyl orange (MO), AgNO3, CuSO4, and CdCl2 were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The double-distilled water was prepared with a Milli-Q water purification system (Milli-Q®Reference, Millipore, Billerica, Massachusetts, USA).

### *2.2. Fabrication of the MoS2 Nanosheets*

The MoS2 nanosheets (MoS2-NS) were prepared through a novel combined method including calcination at high temperature, quenching with liquid nitrogen, and ultrasonic-assisted peeling with hydrazine hydrate. Firstly, 4.000 g of (NH4)2MoS4 was calcinated under nitrogen atmosphere at 800 ◦C for 5 h with a rate of 5 ◦C/min and then a black powder (named bulk MoS2) was obtained. After that, the high-temperature bulk MoS2 was quickly transferred into a Dewar bottle containing liquid nitrogen until the liquid nitrogen gasified completely. Subsequently, the pre-expanded bulk

MoS2 was transferred into a serum bottle with 100 mL of hydrazine hydrate and the bottle was sonicated at a frequency of 40 kHz for 24 h. After centrifugation, the residual MoS2 powders were added into another serum bottle with deionized water and sonicated for 12 h (In addition, the recycled hydrazine hydrate can be reused in a new procedure.). Finally, the resulting suspensions were centrifuged at 3000 rpm for 2 h and then the dark green MoS2-NS dispersions were obtained. After dialyzed with dialysis tubing with 3000 dalton of molecular weight cut off, the obtained ultimate green dispersions were close to 7 of pH and stored in the fridge at 4 ◦C.

### *2.3. Adsorption Batch Experiments*

Adsorption isotherm batch experiments were carried out in a 40-mL serum bottle containing 10 mL liquid with 0.1 g L−1 of the adsorbent concentration. The adsorption isotherm for MB and MO was conducted under 25 ◦C in the range of 0.5 to 50 mg L−1 of the MB and MO concentration and the pH was adjusted to 6.0 ± 0.1 with 1 M H2SO4 solution, while the experiments for heavy metals were conducted under 25 ◦C with metal concentration from 0.5 to 30 mg L−1 and the pH was adjusted to 5.0 ± 0.1 with 1 M H2SO4 solution. After sealed with polytetrafluoroethylene (PTFE) caps, all the bottles were shaken at 250 rpm for 6 h. At sampling points, one bottle was taken out. After filtered with 0.22 μm glass fiber filters (Tianjin Branch billion Lung Experimental Equipment Co., Ltd., Tianjin, China), the MB/MO concentrations were determined by UV-vis spectroscopy (UV-1780, SHIMADZU, Japan) at 664/464 nm, while the residual Cu2+/Cd2+/Ag+ concentrations were analyzed with ICP-MS (XSERIES 2, Thermo). The adsorption isotherms data were treated with Langmuir and Freundlich models [22,23]. The experiments for the adsorption kinetics study were operated at 25 ◦C and 6.0 ± 0.1/5.0 ± 0.1 of pH (adjusted with 1 M H2SO4 solution) in 300 mL dyes/heavy metals solution (20 mg L−1 for dyes and 15 mg L−1 for heavy metals). All the samples were shaken at 250 rpm for 6 h. At sampling points, 1.5 mL of the solution was taken out and then filtered through the filters. The residual dyes/heavy metals concentrations were determined with ICP-MS. The adsorption kinetics data were treated with the pseudo-first order kinetic and pseudo-second-order non-linear kinetic models.

To study the effects of pH values (2–10)/(3–7) on dyes/heavy metals adsorption, the batch experiments were conducted at 25 ◦C in a serum bottle with 20 mg L−1/15 mg L−1 of dyes/heavy metals concentration and 0.10 g L−1 of adsorbents.

### *2.4. Characterization*

The X-ray powder diffraction (XRD) data of the bulk MoS2 and MoS2-NS were tested with X-ray powder diffractometer (MiniFlex600, Rigaku, Milwaukee, Wisconsin, USA) coupled with a Cu *K*α line at 40 kV and 40 mA.

The microstructural features of prepared bulk MoS2 and MoS2-NS were observed with field emission scanning electron microscope (FESEM, Nova NanoSEM 230, FEI, Hillsboro, Oregon, USA), atomic force microscope (AFM, 5500, Agilent USA), and transmission electron microscope (TEM, TECNAI G2F20, FEI, Hillsboro, Oregon, USA).

The Raman data of bulk MoS2 and MoS2-NS were recorded by a confocal laser Raman microscopy (Invia Reflex, Renishaw, UK) with 532 nm of laser wavelength and 0.6 mW of laser energy.

The X-ray photoelectron spectroscopy (XPS) data were recorded with X-ray photoelectron spectrometer (ESCALAB 250, Thermo Scientific, Waltham, Massachusetts, USA) coupled with the Al *Ka* radiation at 15 kV and 51 W. The binding energies were confirmed by using the C1s component as the reference and the binding energy of C-C/H bonds were set at 284.5 eV.

The concentrations of MoS2-NS dispersions were analyzed using UV-vis spectrophotometer (UV-2500, Shimadzu, Japan).

The Brunauer–Emmett–Teller (BET) surface areas of the bulk MoS2 and MoS2-NS were obtained from the analysis of N2-adsorption isotherms at 77 K using the N2 physisorption analyzer (ASAP2020, Micromeritics, Norcross, Georgia, USA).

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


**6.2 Cardiac magnetic resonance imaging**

Multimodality imaging can provide crucial preoperative information, such as a functional and structural assessment of the right ventricle and the tricuspid valve anomaly. This data helps with surgical planning and preoperative counseling. Multimodality imaging can provide personalized details of distinct components of the tricuspid valve. While echocardiography provides correct valvar anatomical details and assessment of right ventricular pressure, cardiac magnetic resonance (CMR) enables a more accurate evaluation of the regurgitant fraction and right ventricular function, which complements the information provided by echocardiography.

Before the cone operation, CMR enables functional and anatomical examination of the RV and tricuspid valve abnormality, which is crucial for surgical planning [30]. Preoperative CMR offered extra information in more than three-quarters of patients, according to a study by Johnson et al., with 69% of the findings changing surgical therapy [31] . Leaflet attachments of the posterior/inferior and anterior leaflets to the RV wall can also be assessed using CMR. There are two types of attachments—focal attachments and linear attachments. Normal attachments, such as focal attachments, allow unobstructed communication between the atrialized and functional RV. Linear attachments occur when the leaflet is completely or partially attached to a muscle shelf at the joint (**Figure 13**).

Additionally, CMR can measure the degree of displacement and rotation of the tricuspid valve and can also measure the Great Ormond Street Hospital score

#### **Figure 14.**

*Cardiac MRI image in four-chamber view showing the calculation of the great Ormond street hospital index using the right atrium and atrialized right ventricle area divided by the sum of the functional right ventricle, left atrium, and left ventricle.*

(**Figure 14**). Most importantly, CMR gives an accurate assessment of chamber size including atrialized and functional right ventricle and RV function. CMR is the gold standard for RV size and function and overcomes the limitations of 2D echocardiography to measure RV size and function.


**1. Introduction**

Rheumatoid arthritis (RA) is a chronic, autoimmune disease, characterized by inflammation and progressive damage of synovial joints, when treated insufficiently [1]. With increasing knowledge about disease pathophysiology, new pharmaceutical strategies and compounds are available. After the application of conventional synthetic diseasemodifying antirheumatic drugs (csDMARDs), such as methotrexate (MTX) in late 1980s, and biological DMARDs since late 1990s, small-molecule Janus kinase inhibitors (JAKis), classified as targeted synthetic DMARDs, represent a new milestone in RA treatment [1–3]. Clinical studies with JAKi demonstrated similar efficacy and safety compared to biological DMARDs [4,5]. However, long-term data for JAKi covering several years are still missing.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases comprising four different types of JAK enzymes in humans: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) [6]. While JAK1, JAK2, and TYK2 are expressed ubiquitously, JAK3 is predominantly detectable in hematopoietic tissue [7,8]. Upon extracellular ligand binding, JAKs associate as homo- or

**Citation:** Reddig, A.; Voss, L.; Guttek, K.; Roggenbuck, D.; Feist, E.; Reinhold, D. Impact of Different JAK Inhibitors and Methotrexate on Lymphocyte Proliferation and DNA Damage. *J. Clin. Med.* **2021**, *10*, 1431. https://doi.org/10.3390/jcm10071431

Academic Editor: Mitsuhiro Takeno

Received: 2 February 2021 Accepted: 29 March 2021 Published: 1 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

heterodimers with type I and type II cytokine receptors. Subsequently, JAK dimers become activated by auto- and transphosphorylation and phosphorylate the cytoplasmic tail of the cytokine receptor [6,8]. This induces the recruitment and binding of signaling molecules, such as the members of the signal transducer and activator of transcription (STAT) family (seven members: STAT1/2/3/4/5A/5B/6). After JAK-mediated STAT phosphorylation, dimerization, and activation, STAT dimers translocate to the nucleus where they act as transcription factors for multiple target genes, modulating, i. a., survival, proliferation, or differentiation of T lymphocytes [9,10]. More than 50 different cytokines and growth factors are known ligands of type I/II cytokine receptors. Depending on the cytoplasmic chains of the receptor, they are able to associate either with only one type of JAK enzyme or with different JAK isoforms. Hence, this creates a high degree of specificity regarding different JAK and STAT combinations [9–11].

Sufficient JAK-STAT signaling is essential in the regulation of immunological processes. Polymorphisms and loss- or gain-of-function mutations within this pathway are associated with immunodeficiency, autoimmune disease, and hematological malignancy [9,12]. Therefore, JAK-targeting agents represent a new class of immunomodulatory drugs [11]. After first approval of JAK inhibiting drugs for the treatment of neoplastic diseases, several JAKi are also authorized for the treatment of RA by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) [13,14]. The two approved first-generation JAK inhibitors tofacitinib (JAK3 > JAK1 > JAK2) and baricitinib (JAK1 > JAK2 > JAK3) are classified as pan-JAK inhibitors, targeting multiple JAK isoforms, but with different affinities. In contrast, the two second-generation JAKi upadacitinib and filgotinib (not yet approved by the FDA) show high selectivity for JAK1 and primarily inhibit its associated cytokine-receptors [10,15]. However, selectivity of JAKi towards specific JAK isoforms is not absolute and depends on dosage and cell type [16]. An overview of reported mean half-maximal inhibitory concentrations (IC50) obtained by enzymatic assays is provided in Table S1.

Although already approved for RA treatment, in vivo and in vitro head-to-head studies of all four JAKi are rare. Therefore, we evaluated the immunomodulatory and cytotoxic potential of tofacitinib, baricitinib, upadacitinib, and filgotinib on human PBMCs freshly isolated from healthy donors. For comparison with conventional synthetic DMARDs, samples treated with MTX were investigated in parallel. JAKi or MTX were either added directly to freshly PHA-stimulated PBMCs or 48 h after PBMC activation, to investigate their impact on preactivated T lymphocytes, as this might be more relevant regarding inflammatory conditions in vivo [17]. Compared to healthy controls, peripheral blood isolated from patients with active RA revealed an enhanced level of activated PBMCs, which may play a direct role in disease pathogenesis [18]. Furthermore, Kitanaga et al. stated constitutive activation of JAK/STAT signaling in PBMCs from patients with systemic sclerosis or RA [19].

JAK/STAT signaling is involved in regulation of multiple fundamental cellular processes. Additionally, there is increasing evidence suggesting that JAK/STAT signaling also modulates molecules involved in DNA damage response pathways [20–24]. To investigate the impact of JAKi on DNA double-strand break (DSB) formation and on repair of radiationinduced DNA damage we quantified nuclear foci stained by γH2AX or 53BP1 (p53-binding protein 1) antibodies. These markers have been described as sensitive molecular indicators for DNA DSBs [25–27].

The objective of the present in vitro study was to compare the immunomodulatory potential of all four JAKi currently approved for RA treatment in Europe. Therefore, we treated freshly and preactivated PBMCs with rising concentrations of either tofacitinib, baricitinib, upadacitinib, filgotinib, and MTX and determined the effect on cell proliferation, activation (CD25) and apoptosis. Furthermore, we investigated the impact of different JAKi and MTX on DNA damage induction and repair by fluorescence microscopic analysis of DNA DSB markers γH2AX and 53BP1. Our study indicates a broad comparability of

the immunomodulatory effects induced by different JAKi and offers a first indication, that (pan)JAKi may impair DNA damage repair in radiated lymphocytes.

#### **2. Experimental Section**

#### *2.1. Ethics Statement*

The study was performed in accordance with the Declaration of Helsinki and was approved by the local ethics committee (No. 183/20). All 14 healthy blood donors (10 female and 4 male; mean age: 35 ± 12 years) who agreed to participate in this study provided written informed consent.


Author details

3.4. Comparison between in vitro cytotoxicity data and in vivo data

only be made by the co-application of various in vivo and in vitro methods.

The importance of cytotoxicity assays in early drug development is unquestionable although it must be concluded that no assay technology for detecting cytotoxicity in vitro is perfect. Strong arguments can be made for and against using cell viability or cytotoxicity assays as a reliable model of human medication. Depending upon the objectives of the investigation, either

4. Conclusion

(right image).

142 Cytotoxicity

As the whole, medical science and industry is based on the modification, repair of damaged or badly functioning cells and tissues in human or animal body, the correlation between in vitro and in vivo data is crucial. Do these artificial test systems, cell lines truly replicate how a real tissue would react to a certain treatment or compound? The answer is based on the application of multiple in vitro methods and the careful planning of the in vivo experiments. A good example of the practice is the study of Yu et al. [37]. Xantii fructus is a traditional Chinese herbal drug and clinical reports indicated its renal toxicity. The study was based on MTT and LDH assays of the main components of the herbal drug on a renal cell line, as well as acute and chronic toxicity experiments in rats. While the main component of the drug, the atractyloside potassium salt showed no cytotoxicity on the cell lines, the water extract of the fruit had an inhibitory effect in case of high concentrations on the MTT assay, but no membrane damage on the LDH assay. These results indicate that the secondary components of the water extract have cytotoxic capabilities and the exact mechanism of killing might involve the suppressed metabolic activity of the cells, but not the damage of the cell membrane. The acute in vivo toxicity showed that only high concentrations could terminate the rats and cause abnormalities in the organs and the chronic toxicity showed only minor changes in the highest concentration group. Overall, this complex study created a much more accurate, scientific point of view about the toxicity of Xanthii fructus, what chemicals are responsible for its toxicity, what are the exact dosages, and what are the side effects that are caused by the herbal drug. It could

Figure 10. Chemical formula of the Calcein-AM (upper image), Hoechst 33342 (left image) and the propidium iodide

Ildikó Bácskay\*, Dániel Nemes, Ferenc Fenyvesi, Judit Váradi, Gábor Vasvári, Pálma Fehér, Miklós Vecsernyés and Zoltán Ujhelyi

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary


**4. Improved Islanding Detection Method Based on the Proposed Solution**

According to the previous section, the cancellation problem occurs by using two perturbations in the opposite directions. The main meaning of the solution is how to prevent the occurrence of cancellation.

First, the beginning of the proposed IDM uses the perturbation signal in the range *n* = 0–1.1. As the range of perturbation signal includes values over and below the normal value (*n* = 1), the injected signal cancellation caused by the perturbation signals in opposite directions can occur.

Second, to prevent the occurrence of cancellation, the perturbation signal value is changed so that the opposite signals cannot occur.

Therefore, the solution for the cancellation problem is to change the perturbation signal values in the range *n* = 0–1.1 to *n* = 0–1, and in the normal condition from *n* = 0.9–1.1 to *n* = 0.9–1. By changing this value, the injected signal cancellation is eliminated. Similar to the Equations (3)–(15), the solution is explained below:

The injected signal cancellation occurs when:

$$2\mathfrak{n}\_0 = \mathfrak{n}\_1 + \mathfrak{n}\_2 \tag{16}$$

By using the new perturbation signal (*n*0 = 1, *n*1 = 1, and *n*2 = 0.9), Equation (16) becomes

$$2 \neq 1.9\tag{17}$$

Based on Equation (17), the fluctuation of DC-link voltage caused by the perturbation signals from PV1 and PV2 cannot cancel each other. Thus, the proposed solution can solve the injected signal cancellation.

*Appl. Sci.* **2019**, *9*, 4054

By generalizing this solution with *nk* perturbation signals (*k* = 1 ÷ ∞), the injected signal cancellation occurs when:

$$
\Delta v\_{1d\mathcal{c}}(t) + \Delta v\_{2d\mathcal{c}}(t) + \dots + \Delta v\_{kd\mathcal{c}}(t) = 0 \tag{18}
$$

or

$$n\_0 - n\_1 + n\_0 - n\_2 + \dots + n\_0 - n\_k = 0\tag{19}$$

Finally,

$$kn\_0 = \sum\_{k=1}^{\infty} n\_k \tag{20}$$

As *n*0 = 1 (without perturbation signal) and *nk* ≤ 1, Equation (20) is correct when all perturbation signal values are 1 (without perturbation signal case).

With other values of the perturbation signals (with perturbation signal), Equation (20) becomes

$$k n\_0 > \sum\_{k=1}^{\infty} n\_k \tag{21}$$

For this reason, the injected signal cancellation is eliminated by using the proposed solution.

The flowchart in Figure 7 explains the detailed procedure of the proposed solution. The solution is verified by a step-by-step simulation.

**Figure 7.** Flowchart of the improved IDM program (*n*0 is the perturbation factor at the beginning, ∆*V* is the voltage deviation, *Eps* is the abnormal event value, *dP* is the rate of change of output power).

The testing scenarios in Table 3 are the worst case (the power of DGs and the load are balance), and the procedure is similar to the previous testing procedure.

**Table 3.** Multi-PV operation scenario.

The model parameters are almost similar to the previous testing, only the new perturbation factors are different. The perturbation factors at normal condition in cancellation case are *n* = 1–0.9 for PV1 and *n* = 0.9–1 for PV2.

In addition, the eight-PV scenario also tests to verify the effect of the improved IDM. The parameters of the eight-PV scenario are as below:

As shown in the results in Figure 8, the injected signal cancellation occurs in two-PV scenario. With the improved islanding detection method based on the proposed solution, the islanding condition is detected after 40 ms when the islanding occurs. The solution is effective in two-PV scenario.

**Figure 8.** Solution result in two-PV scenario.

The results of three-PV scenario are shown in Figure 9 and 10. In Figure 9, the islanding detection method can not detect the islanding phenomenon when injected signal cancellation occurs.

The results in Figure 10 show that the islanding condition is detected after 64 ms when the islanding occurs by applying the proposed solution to the improved islanding detection method in the injected signal cancellation case.

The four-PV scenario has the similar results, the islanding condition can be detected after 48 ms when the islanding occurs by using the improved islanding detection method, but it cannot be detected by using the original one. The results are shown in Figures 11 and 12.

**Figure 11.** Cancellation result in four-PV scenario.

**Figure 12.** Solution result in four-PV scenario.

The eight-PV scenario result is shown in Figure 13. The improved islanding detection method by applying the proposed solution can detect the islanding condition after 48 ms.

**Figure 13.** Solution result in eight-PV scenario.

Based on the simulation results and mathematical explanations, the proposed solution eliminates the injected signal cancellation by using the improved islanding detection method based on the proposed solution in two-PV, three-PV, four-PV, and eight-PV scenarios.

Moreover, the injected signal cancellation is eliminated not only in the specified cases but also in the general case by using the improved islanding detection method. This result shows the achievement and efficiency of the proposed solution.


**3. Chitosan-Based Nanoparticles**

Chitosan is a polysaccharide, with 2-deoxy-2-(acetylamino) glucose units bonded by 1,4-glycosidic linkages; it is prepared by partial N-deacetylation of chitin (Figure 3). Chitin has good mechanical strength, is biocompatible, bioactive and biodegradable, but has limited utilization due to low solubility. Therefore, it is converted into chitosan by deacetylation in the presence of hydroxide at high temperatures [38]. Chitin is extracted from marine organisms such as lobsters and molluscs, and from crab shells, insects, yeast, and fungi. A long polymeric chain is composed of glycosidic linkages. Chitosan is insoluble in sulfuric acid and phosphoric acid but soluble in organic solutions with a pH lower than 6.5, such as acetic acid, citric acid, and tartaric acid. Due to its solubility, chitosan is present in films, hydrogels, pastes, nanoparticles, and nanofilms [39,40]. Chitosan has a greater degree of deacetylation and molecular weight; the variation in the size of a particle and aggregation is dependent upon the degree of deacetylation and molecular weight [3]. Recently, chitosan and its derivatives have been considered as the best vehicle in the pharmaceutical field due to their biocompatibility, and their non-carcinogenic, non-toxic, antibacterial properties. Chitosan offers a large range of options for industries and scientists for the generation of modified and novel drug delivery systems. Chitosan acts as an auxiliary agent in the therapeutic application for tissue engineering, wound dressing, and sliming. Protonation of the amino group in an acidic medium results in cation formation. Chitosan exhibits unique behavior because of its cationic nature [38,41]. Modification of chitosan and the stability of drugs delivered using chitosan decrease the adverse effects of diseases and increase the biocompatibility of drugs for various diseases [42].

**Figure 3.** Structure of chitosan.

Chitosan nanoparticles widely act as a potential carrier for therapeutic applications. The cationic nature of chitosan is useful for the development of a drug delivery system. One of the major benefits of chitosan nanoparticles is their rapid uptake across the cell membrane due to the presence of amine groups. Complexation of chitosan with anionic charged polymers results in an interesting gelation property [43]. Methods generally applied for the preparation of chitosan nanoparticles include the microemulsion method, ionic gelation, and micro-emulsion solvent diffusion method [3,38]. In the microemulsion method, chitosan nanoparticles are prepared, and micellar droplets are cross-linked in the presence of glutaraldehyde. Surfactant (hexane mixture) is added to the chitosan acetic/glutaraldehyde solution. Surfactant helps in the formation of chitosan nanoparticles smaller than 100 nm under continuous stirring to complete cross-linking between the amine group of chitosan with glutaraldehyde. By applying a low pressure, the excess organic solvent is removed. The excess organic solvent used, the complexity of the washing process, and the amount of time this method takes is its main drawback [3,44]. Chitosan nanoparticles prepared using the inotropic gelation method depend upon electrostatic interaction between the amino group of chitosan and the polyanions group such as triphosphate in an aqueous medium [45–48]. Firstly, the chitosan is dissolved in acetic acid in the presence of stabilizers such as poloxamer. So, nanoparticles are formed under continuous stirring. The size of the nanoparticle depends upon the ratio of chitosan-to-stabilizer [3]. The emulsion solvent diffusion method is based on the addition of an organic phase into a chitosan solution containing a stabilizer at a higher temperature and pressure with constant stirring. Nanoparticles are formed by the addition of water to an organic solvent. This method of nanoparticle formation is better for hydrophobic-based drug delivery systems. This method also has some drawbacks, such as greater shear force and harsh process conditions [49]. Chitosan is one of the most extensively studied biopolymers because chitosan possesses some ideal properties for polymeric carriers for nanoparticles. The properties are given in Table 1.

Current studies have shown a massive variation of applications and novel modifications of chitosan, thereby increasing its overall value. Drug encapsulation using chitosan with tripolyphosphate was first reported by Bodmeier et al. [50,51] who used an inotropic gelation process, which results in the formation of chitosan nanoparticles. Chitosan (100–150 nm) nanoparticles were loaded with Rosuvastatin drug encapsulated in polyvinyl alcohol (PVA)/Sodium alginate (SA) core using an ionic gelation method. The release behavior of the drug was observed within 24 h, and chitosan particles delivered significant results in the drug release process. This biocompatible and biodegradable drug delivery system was a suitable choice for replacing the various doses of the drug Rosuvastatin [51]. In 1998, Alonso et al. synthesized the chitosan nanoparticle with the development of inter-and intra-molecular interaction between the chitosan amino group and tripolyphosphate (TPP). This method produced a high yield of chitosan nanoparticles [43]. Thandapani Gomathi et al., in 2017, synthesized chitosan nanoparticles with TPP-loaded drugs with letrozole (LTZ) for cancer treatment. Characterization techniques such as SEM, TEM, FTIR, XRD, and TGA results showed the optimum results. Additionally, the prepared formulation was evaluated in vitro to determine its biodegradability, hemocompatibility, and serum stability. The preliminary studies supported the assertion that chitosan nanoparticle synthesis had

biocompatibility and hemocompatible applications, and could act as an essential pharmaceutical excipient for letrozole [52]. Synthesis techniques for chitosan nanoparticles are given in Figure 4.

**Table 1.** Some ideal properties of polymeric nanocarriers [3].


**Author details**

Anika Farzin1 \*, Md. Mizanur Rahman2 and Ferdows Ara Mollika3

1 Department of Microbiology, Eastern Medical College, Dhaka, Bangladesh

2 Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet, Bangladesh

3 Department of Microbiology, Tairunnessa Memorial Medical College, Gazipur, Bangladesh

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.