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Q1:ments ons Button rations A https://uab.instructure.com/groups/346802/discussion_topics/7329486 pasting it into the reply area of the discussion. De sure to come back and respond to your group members. ib S R Description: UAB Hospital has decided to purchase a unique and very expensive type of MRI scanning equipment (to detect tumors). Look up some information on the internet about what is MRI equipment and how would it be used by a health care provider. The General Electric Company (GE) is a major provider of MRI equipment. It is important for the sales manager of GE Healthcare to understand who is likely to be involved in the buying center for this purchase at UAB Hospital. Each person may influence the purchase and perhaps influence it in different ways. The sales manager needs to see if all of the different buyer center's needs are being met, and if not, why not. Clearly, this kind of thinking can help guide the sales strategy. See the section in the book about "Multiple buying influence in a buying center" for more discussion of the buying center concept. INITIAL POST: In a few sentences, introduce yourself to your new discussion group. . Then address these points related to the case above: . Who might be involved in the buying center? • Explain your answer and describe the type of influence that different people in the buying center at UAB Hospital would have on the purchase.See Answer
Q2:1. The following questions pertains to the breakdown of glucose for ATP. (15 points) a. What is Glucose? What is Glycogen? b. How many ATP molecules are produced during glycolysis per glucose molecule? Does this ATP production require oxygen? c. Why do muscle cells produce lactic acid during bursts of activity? d. How many ATPs are produced per glucose molecule in the mitochondria during oxidative phosphorylation? Does this ATP production require oxygen? e. Why is there a membrane potential across the inner mitochondrial membrane?See Answer
Q3:6. The action potential on a motor neuron lasts about 1.6 ms. A large motor neuron conducts the action potential at about 200 m/s. (10 points) a. Calculate the distance between the beginning of the action potential and its end. b. The nodes of Ranvier are interruptions in the myelin sheath in myelinated fibers, and typically they are located about 2 mm apart along the length of the axon. The action potential that you calculated in Part A is spread out over how many nodes of Ranvier? c. In Saltatory conduction, what "jumps" from node to node?See Answer
Q4: Individual Assignment – Mini Literature Review (700-900 words) (20% of final module mark) This report will be a literature review on a research topic of interest covered in the 'Biomaterials' classes. General guidelines: The purpose of this review is to give a brief overview of the studies performed on the selected topic and to provide a critical evaluation of the literature in a clear and concise manner. It should be an original piece of work. Only peer review journal experimental articles should be included in the review, which should be clearly cited. Students should not use other review papers to form the basis of their review. The overall objective of the assignment is to familiarise students with the scientific literature, understand these articles and critique their content. A good review identifies what information is important and condenses that information for the reader. Better understanding of the subject will make it easier to explain it thoroughly and briefly. A minimum of 8 experimental scientific papers should be used and referenced in the review (no need to submit these papers with your report). This mini-review should be between 700-900 words (excluding references). The review should: be written in your own words; contain the main arguments and critical evaluation of the papers; • be clearly structured, have an introduction and a conclusion; • • indicate where you agree or disagree with the authors. The review should not: use other review papers published in the literature as the basis of this report; • • be produced using cut-and-paste at any stage; . contain any sentences (or phrases more than a few words long) occurring in the original paper (quoted or not). Provide all references used at the end of your report. Use an appropriate referencing style, for example the ACS style (link given below) or a different one: https://pubs.acs.org/doi/full/10.1021/acsguide.40303See Answer
Q5: Research Methodology Spring 2023- 2024 Assignment on "Materials and Methods" Let's examine the "Materials and Methods" sections of the published papers that you chose. How are they similar and different?? You need to select two different papers addressing similar problems. Select one common experiment that was done in both papers, and compare between the two papers on the similarities and differences of this experiment using the below form. 13 > - + ** Paper 1 Title: Link: Materials and Methods of the experiment you selected: Paper 2 Title: Link: Materials and Methods: Materials and Methods of the experiment you selected: Table 1: Differences in materials and methods of your selected experiment between Paper 1 and Paper 2. Paper 1 Paper 2 Table 2: Similarities in materials and methodology of your selected experiment between Paper 1 and Paper 2. + + Your feedback/ opinionSee Answer
Q6:11:53 < CHE3008 Individual Assig... Q L 5G 62 Individual Assignment - Mini Literature Review (700-900 words) (20% of final module mark) This report will be a literature review on a research topic of interest covered in the 'Biomaterials' classes. Suomasion dreadme General guidelines: The purpose of this review is to give a brief overview of the studies performed on the selected topic and to provide a critical evaluation of the literature in a clear and concise manner. It should be an original piece of work. Only peer review journal experimental articles should be included in the review, which should be clearly cited. Students should not use other review papers to form the basis of their review. The overall objective of the assignment is to familiarise students with the scientific literature, understand these articles and critique their content. A good review identifies what information is important and condenses that information for the reader. Better understanding of the subject will make it easier to explain it thoroughly and briefly. A minimum of 8 experimental scientific papers should be used and referenced in the review (no need to submit these papers with your report). This mini-review should be between 700-900 words (excluding references). The review should: ⚫be written in your own words; ⚫be clearly structured, have an introduction and a conclusion; contain the main arguments and critical evaluation of the papers; ⚫ indicate where you agree or disagree with the authors. The review should not. ⚫use other review papers published in the literature as the basis of this report; be produced using cut-and-paste at any stage; ⚫contain any sentences (or phrases more than a few words long) occurring in the original paper (quoted or not). Provide all references used at the end of your report. Use an appropriate referencing style, for example the ACS style (link given below) or a different one: https://pubs.acs.org/doi/full/10.1021/acsguide.40303 2 35 Dashboard Calendar To-do Notifications InboxSee Answer
Q7: Dovepress INTERNATIONAL JOURNAL OF NANOMEDICINE OPEN ACCESS International Journal of Nanomedicine Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering a ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/dijn20 Li Chen, Jiaxin Liu, Ming Guan, Tongqing Zhou, Xin Duan & Zhou Xiang To cite this article: Li Chen, Jiaxin Liu, Ming Guan, Tongqing Zhou, Xin Duan & Zhou Xiang (2020) Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering, International Journal of Nanomedicine,, 6097-6111, DOI: 10.2147/IJN.S249829 To link to this article: https://doi.org/10.2147/IJN.S249829 CrossMark © 2020 Chen et al. Published online: 14 Aug 2020. bmit your article to this journal Article views: 433 QView related articles View Crossmark data Citing articles: 61 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=dijn20 Taylor & Francis Taylor & Francis Group International Journal of Nanomedicine Open Access Full Text Article Growth Factor and Its Polymer Scaffold-Based Delivery System for Cartilage Tissue Engineering 1,2,* Li Chen Jiaxin Liu¹›* Ming Guan 2,3 Tongqing Zhou² Xin Duan' I Zhou Xiang 'Department of Orthopedics, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China; 2School of Dentistry, University of Michigan, Ann Arbor, MI, 48109, USA; ³Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China *These authors contributed equally to this work Correspondence: Xin Duan Tel +86-28-85422426 Email dxbaal@hotmail.com Zhou Xiang Tel +86-28-85422605 Email xiangzhou15@hotmail.com submit your manuscript | www.dovepress.com DovePress in http://doi.org/10.2147/1JN.S249829 This article was published in the following Dove Press journal: International Journal of Nanomedicine Dovepress open access to scientific and medical research REVIEW Abstract: The development of biomaterials, stem cells and bioactive factors has led to cartilage tissue engineering becoming a promising tactic to repair cartilage defects. Various polymer three-dimensional scaffolds that provide an extracellular matrix (ECM) mimicking environment play an important role in promoting cartilage regeneration. In addition, numer- ous growth factors have been found in the regenerative process. However, it has been elucidated that the uncontrolled delivery of these factors cannot fully exert regenerative potential and can also elicit undesired side effects. Considering the complexity of the ECM, neither scaffolds nor growth factors can independently obtain successful outcomes in carti- lage tissue engineering. Therefore, collectively, an appropriate combination of growth factors and scaffolds have great potential to promote cartilage repair effectively; this approach has become an area of considerable interest in recent investigations. Of late, an increasing trend was observed in cartilage tissue engineering towards this combination to develop a controlled delivery system that provides adequate physical support for neo-cartilage formation and also enables spatiotemporally delivery of growth factors to precisely and fully exert their chon- drogenic potential. This review will discuss the role of polymer scaffolds and various growth factors involved in cartilage tissue engineering. Several growth factor delivery strategies based on the polymer scaffolds will also be discussed, with examples from recent studies highlighting the importance of spatiotemporal strategies for the controlled delivery of single or multiple growth factors in cartilage tissue engineering applications. Keywords: polymer scaffold, growth factor, delivery, cartilage repair Introduction Articular cartilage is a specific type of connective tissue that covers the articular surfaces of the bone; it is mainly composed of a dense extracellular matrix (ECM) and a sparse cell population. It plays an essential role in the biomechanical functions of the joints, including shock absorption, sheer resistance and load bearing.² Once articular cartilage is damaged, it has a limited potential for sponta- neous repair due to the lack of vascularity, nerves and lymphatics. This can result in joint pain, swelling, dysfunction, and eventually lead to osteoarthritis (OA).³,4 In the past two decades, OA has been the most common form of arthritis accounting for approximately 300 million patients worldwide and undoubtedly has been considered as one of the most significant health problems that also pose a substantial financial burden on the public health system, and the patients themselves. Currently, conservative treatments, including pharmacological and non-pharmacological therapies, are commonly applied to improve joint pain, reduce stiffness and improve physical function in patients with OA. However, the therapies сс International Journal of Nanomedicine 2020:15 6097-6111 02 Ⓒ2020 Chen et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php NC and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). 6097 Chen et al 7-10 cannot prevent further joint degeneration. Surgical strate- gies are also designed to treat cartilage defects, including autologous chondrocyte implantation (ACI), microfrac- ture, osteochondral grafts and total joint arthroplasty. However, they have some inherent shortcomings such as the requirement of secondary surgery, immunogenic responses, shortage of donor tissues and pathogen trans- mission risks. Alternatively, cartilage tissue engineer- ing, which involves combining cells, scaffolds and growth factors, has emerged as a promising strategy for cartilage repair.¹¹ Meanwhile, the methods of minimally invasive surgery, including the implantation and injection, are an important component of clinical translation of tissue engi- neering techniques which have been verified in vitro. ¹2 In general, scaffolds as biologically active ECM provide mechanical support for cell growth and chondrogenic dif- ferentiation, which could be beneficial for stimulating and accelerating the cartilage regeneration process. With the development of chemistry and processing, numerous synthesized and natural materials have been applied to fabricate scaffolds that successfully promote the cartilage regeneration without noticeable signs of immune response and rejection. ¹3-15 12 16,17 While biomimetic three-dimensional scaffolds have been made, they cannot create high-quality cartilage tissue independently. Stem cells, pluripotent cells and native progenitor cells are commonly used in combination with scaffolds to accelerate and improve the regeneration process. Moreover, cell-based therapies are influenced by the cellular microenvironment to some extent. Growth factors are of high importance as they have the potency to induce and enhance cellular responses, which is beneficial for the cells as they need to differentiate into desired lineages.¹8 Although scaffolds can obtain sufficient growth factors from the culture medium under in vitro conditions, the incorporated growth factors can spread out of the scaffolds and degrade in a short time in vivo. Besides, different dosages and delivery rates are required for dif- ferent growth factors to induce the cells in in vitro or in vivo conditions. Today, a plethora of studies have been conducted to investigate the delivery of single or multiple growth factors from the scaffolds in a defined 18 19,20 manner. This review examined the delivery of growth factors for cartilage tissue engineering, with an emphasis on the polymer scaffold-based approaches. First, the aim is to enable an understanding of current applications of polymer scaffolds, following with the descriptions of different 6098 submit your manuscript | www.dovepress.com DovePress Dovepress growth factors involved in cartilage tissue engineering. A latter section will place a particular emphasis on the growth factor delivery strategies associated with polymer scaffolds. Finally, the current challenges and suggestions of polymer scaffold-based growth factor delivery for car- tilage tissue engineering are explained. Polymer Scaffolds Articular cartilage, with its unique mechanical properties pro- vides the contact surfaces for load transfer between bones, which enables the joint to withstand weight-bearing. The ability to do so is attributed to its complex structure comprised of a fluid phase and a solid matrix that is composed mainly of a depth-dependent collagen fibrous network and proteogly- cans, as well as other types of proteins, lipids, and cells. Therefore, the scaffold suitable for cartilage tissue engineering should have good biocompatibility for cell adhesion, migra- tion and proliferation, and also provide appropriate mechanical and structural support. In addition, biodegradability and being free of adverse reactions are basic properties required for a three-dimensional scaffold mimicking physiological characteristics.2¹ Currently, a wide range of natural and syn- thetic polymers play an important role in the development of scaffolds for cartilage tissue engineering. Due to superior biocompatibility and biodegradation, natural polymers like collagen, chitosan, silk fibroin, alginate, hyaluronic acid and chondroitin sulfate are suitable for initiating a fast regeneration process. However, potential pathogen transmission, immuno- genicity and poor mechanical properties limit their clinical application. On the other hand; synthesized polymers can artificially regulate the degree of polymerization, thereby con- trolling its mechanical properties, internal structure and degra- dation, which can effectively promote the regeneration process. Poly (lactic acid) (PLA), poly glycolic acid (PGA), poly lactide-co-glycolic acid (PLGA) and poly caprolactone (PCL) are the most commonly synthesized polymers in the application of three-dimensional scaffolds for cartilage tissue engineering.23 When comparing these to natural polymers, the properties of synthetic polymer-based scaffolds are consider- ably different in terms of their tunable properties, such as molecular weight, transition temperatures and crystallinity.24 Polymer nanofibers have been extensively studied due to their ability to encapsulate and deliver growth factors for different tissue regeneration purposes. Nanofiber scaf- folds with high surface to volume ratio and interconnected porous structure, seem to hold the lead position as the ideal candidate for cartilage tissue engineering. They play a role in stimulating the ECM environment, allowing 21 International Journal of Nanomedicine 2020:15 Dovepress cells to populate empty spaces and organize themselves, and mechanical stimulation can be applied to this porous structure to orient the cells and maintain a chondrocyte phenotype. As a result, scaffolds will be degraded and replaced by newly formed ECM, without producing adverse effects due to the degradation products. To date, various technologies such as electrospinning, phase separation, self-assembly, drawing and template synthesis have been applied in attempts to optimize nanofiber scaf- folds to make them more consistently bioactive and mechanically stable for effective tissue regeneration application. For example; a nanofibrous scaffold was developed that was highly porous, interconnected and degradable. It was developed using phase separation of poly l-lactic acid (PLLA) solutions combined with poro- gen leaching techniques. Through a series of characteristic tests, chondrogenic evaluations in vitro and in vivo demonstrated that this nanofibrous PLLA scaffold is an excellent candidate providing an advantageous three- dimensional microenvironment for a wide variety of car- tilage repair strategies (Figure 1).² 27-29 Growth Factors 30 Growth factors are a group of peptides that mediate cel- lular proliferation, migration and differentiation by bind- ing to transmembrane receptors located on target cells. When a sufficient number of receptors are activated, the signaling transduction process may trigger a series of specific cellular activities.³0 Concerning cartilage develop- ment, growth factors play an essential role in regulating the processes of chondrogenesis and hypertrophy, such as the members of transforming growth factor-ß (TGF-B) superfamily, insulin-like growth factor-1 (IGF-1), fibro- blast growth factor (FGF) family and platelet-derived growth factor (PDGF). In order to provide a better under- standing of their potential, descriptions of their roles involved in the regeneration and maintenance of articular cartilage will now be described (Table 1). Transforming Growth Factor-B Superfamily The TGF-ß superfamily is comprised of more than 30 closely related polypeptides, mainly including typical TGF-ßs, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and activin/inhibin, which regulate multiple cell functions from early develop- ment to regulating homeostasis throughout adult life.³1 31 International Journal of Nanomedicine 2020:15 Chen et al A large number of studies have shown that they have significant regulatory effects on the homeostasis and repair of articular cartilage. Transforming Growth Factor-B TGF-B is a dimer with a molecular weight of 25 kilo Daltons (kDa) that is composed of two identical or similar chains. There are three isoforms (1-3) that are generally considered to be potent stimulators in all stages of chon- drogenesis with a function of inducing proteoglycans and type II collagen synthesis. ³2 TGF-ß signaling transduction is based on the membrane-bound heteromeric receptors (type I and type II). Binding to type II receptors leads to the phosphorylation of type I receptors, causing the phos- phorylation of TGF-B specific Smad proteins, particularly Smad 2 and 3. In addition, some Smad-independent pathways, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (Erk) and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) can also be activated by TGF-B.³4 33 34 35 TGF-B is one of the main initiators of chondrogenesis of mesenchymal precursor cells, and the differentiation of mesenchymal stem cells (MSC) into chondrocytes also requires its stimulation. The expression of N-cadherin was induced by strong stimulation of TGF-ß to enhance cell adhesion and aggregation, and subsequently promote cell proliferation, differentiation and deposition of the cartilage- specific extracellular matrix.³5 Among these three isoforms, TGF-B1 was the first to be discovered, and TGF-ß1 and TGF- 33 have been used in a large number of studies to explore the effect of TGF-B on the repair of cartilage after it defects. Although some studies suggest that the ability of TGF-32 and TGF-33 to promote cartilage differentiation may be more superior to that of TGF-31, there is a consensus that there is no significant difference among the three TGF-B isoforms regarding their ability to promote cartilage differentiation. 36,37 In a Sprague-Dawley rat full-thickness cartilage defect model, Lentivirus-TGF-ß1-EGFP transduced BMSCs/calcium alginate gel significantly improved the amount of glycosaminoglycan (GAG) and type II collagen in the defect area in the early stage via activating the Smad pathway, when compared to a BMSCs/calcium alginate gel without TGF-31 transfection. Hypertrophy markers gene expression of chondrocytes were also inhibited by increasing Yes-associated protein-1 (YAP-1).38 Additionally, TGF- 31-incorporated collagen vitrigel had a better effect on mana- ging the early pain mitigation and osteochondral defect repair compared to collagen vitrigel alone.39 Moreover, BMSC submit your manuscript | www.dovepress.com DovePress 6099 Chen et al A Acc. V Magn 10.0kV 100x C 6100 E 200m were induced for the first 4 days with transient soluble TGF- B1, in which the accumulation of proteoglycans was 10-fold higher than TGF-B1-free culture after 3 weeks. These results suggest that TGF-ß promotes chondrogenic differentiation mainly depends on the extent of stimulation of the first week. 40 Nevertheless, there are still some studies that do not support the role of TGF-ß in cartilage repair in vivo. In a rabbit osteochondral defect model, oligo polyethene glycol B D submit your manuscript | www.dovepress.com DovePress F Figure I Nanofibrous PLLA scaffolds induce cartilage regeneration in vitro and in vivo. (A) SEM micrographs of nanofibrous PLLA scaffolds with macro-porous structures (Scale bar: 200 µm). (B) SEM micrographs of the nanofibrous microstructure of the pore walls at a higher magnification (Scale bar: 10 µm). (C) H&E staining showed that BMSCs grew throughout the whole scaffolds after 4 weeks in vitro chondrogenic culture on nanofibrous PLLA scaffold (Scale bar: 200um). (D) Alcian blue staining showed a dense GAG matrix deposition after 4 weeks in vitro chondrogenic culture on nanofibrous PLLA scaffold (Scale bar: 100 µm). (E) H&E staining revealed that BMSCs/ nanofibrous PLLA scaffold constructs had typical cartilage morphology after 8 weeks implanted in nude mice (Scale bar: 200 µm). (F) Safranin-O staining showed that BMSCs/nanofibrous PLLA scaffold constructs were positive for GAG-containing matrix in vivo (Scale bar: 200 µm). 10 mm. Notes: Reprinted from Gupte MJ, Swanson WB, Hu J, et al. Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization. Acta Biomater. 2018;82:1-11. Copyright (2018), with permission from Elsevier.27 Dovepress (PEG) fumarate (OPF) hydrogel composites containing gela- tin microparticles (GMPs) loaded with MSCs with or without TGF-B1 did not improve cartilage morphology. Besides, undesirable side effects such as synovial fibrosis, endochon- dral ossification and hypertrophic scars were observed in vivo after a continuous stimulation by TGF-B1.4 Therefore, it is crucial to properly deliver and present TGF- ẞs in vivo for cartilage regeneration. 42-44 International Journal of Nanomedicine 2020:15See Answer
Q8: Downloaded via UNIV OF SHARJAH on February 25, 2024 at 21:05:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACS! Biomaterials SCIENCE & ENGINEERING pubs.acs.org/journal/abseba Engineering a Cortisol Sensing Enteric Probiotic Vaughn Litteral,* Rebecca Migliozzi, David Metzger, Craig McPherson, and Roland Saldanha Cite This: ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 Metrics & More ACCESSI ABSTRACT: Chronic stress can lead to prolonged adrenal gland secretion of cortisol, resulting in human ailments such as anxiety, post-traumatic stress disorder, metabolic syndrome, diabetes, immunosuppression, and cardiomyopathy. Real time monitoring of chronic increases in cortisol and intervening therapies to minimize the physiological effects of stress would be beneficial to prevent these endocrine related illnesses. Gut microbiota have shown the ability to secrete, respond, and even regulate endocrine hormones. One such microbe, Clostridium scindens, responds transcriptionally to cortisol. We engineered these cortisol responsive genetic elements from C. scindens into an enteric probiotic, E. coli Nissle 1917, to drive the expression of a fluorescent reporter allowing for the designing, testing, and building of a robust and physiologically relevant novel cortisol probiotic sensor. This smart probiotic was further engineered to be more sensitive and to respond to elevated cortisol by expressing tryptophan decarboxylase, thereby bestowing the ability to generate tryptamine and serotonin. Here we show that upon cortisol treatment the smart probiotic produces measurable amounts of tryptamine. Accumulated levels of these neuromodulators should improve mood, anxiety, and depression and drive down cortisol levels. Importantly, this work can serve as a model for the engineering of a sense-and-respond probiotic to modulate the gut-brain axis. KEYWORDS: engineered probiotic, probiotics, smart probiotic, gut brain axis, Escherichia coli Nissle 1917, EcN, cortisol, glucocorticoid, 5-alpha-tetrahydrocortisol, adrenal, chronic stress, tryptophan, tryptophan decarboxylase, 5-HT, serotonin, trypta lysR transcription factor, bacterial sensor, Clostridium scindens, bacterial targeted gene integration, biosensor, sense and respond circuit INTRODUCTION The prevalence of anxiety, stress, and depression is ever increasing in modern societies. Mitigating the endocrine response in times of chronic anxiety, stress, and depression has historically been a challenge due to the endocrine system's intimate role in physiological homeostasis. Neuman et al.² demonstrated that endogenous microbial sentinels have been shown to both monitor and influence the endocrine system and ultimately human physiology. Probiotics show promise in restoring homeostasis in endocrine and immunological dysregulation. More recently, engineering probiotics as living diagnostics and therapeutics has allowed for a whole cell therapeutic approach with a beneficial metabolic potential that seems unlimited. Decades of research into the interplay between the gastrointestinal tract and the nervous system referred to as the gut brain axis (GBA) has revealed an intricate communication network shown to have a great impact in human health, development, and physiology. The GBA is a bidirectional communication system between the nervous system (central nervous system, autonomic nervous system, and enteric nervous system), and hypothalamus pituitary adrenal (HPA) axis with gut microbiota symbionts. Sudo et al. demonstrated that microbiota were essential for HPA develop- ACS Publications © 2023 The Authors. Published by American Chemical Society Read Online Article Recommendations 5163 T SI Supporting Information E. coli Nissle 1917 Ato/C:lysRtdc Chromosomal DNA lysR tryptophan tdc Received: November 1, 2022 Accepted: April 17, 2023 Published: August 30, 2023 LysR tryptophan decarboxylase (TDC) Article an tryptamine. Special Issue: Design and Evaluation of Engineered Probiotics Cortisol ment in germ free mice. Stressors, such as from the host inflammatory response and environmental factors, activate the HPA through the hippocampus release of CRF (corticotropin releasing factor), which in turn stimulates the pituitary to release ACTH (adrenal corticotropic hormone). ACTH stimulates specific cellular receptors, resulting in adrenal secretion of the stress hormone cortisol. While acute stress can be beneficial, chronic stress stimuli can lead to chronic cortisol secretion with detriments to human health and performance (i.e., anxiety, stress, post-traumatic stress disorder, diabetes, immunosuppression, cardiomyopathy, weight gain, and alcoholism). About 10% of cortisol is in active form and present in the blood circulation, whereas the remainder is bound to albumin or cortisol binding globulin protein. Cortisol and associated glucocorticoid metabolites regulate nearly every cell type in the body through the glucocorticoid 15 TOIC SACS materials https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 16 and mineralocorticoid receptors acting as seemingly global transcription factors in gene expression. Traditional ther- apeutic interventions into ameliorating the endocrine response is met with many adverse events in off target tissues as cortisol exerts this global but tissue specific gene expression response. ¹7 18-20 Noninvasive monitoring of free cortisol in sweat and saliva has evolved in recent years as an interest in the human performance and health monitoring industry as well as the United States Department of Defense. Cortisol in saliva, sweat and tears is indicative of blood levels and is a predictive biomarker in determining real time psychological stress.ª Gastro-intestinal glucocorticoid levels have been difficult to ascertain, wherein bacterial secondary metabolism of cortisol and resultant desmolysis occurs beginning in the large intestine. Ridlon et al. employed RNA-seq to investigate cortisol metabolism and its regulated gene expression in one such gut bacterium, Clostridium scindens. C. scindens demon- strated robust response in gene expression to the stress hormone, cortisol, and observed side chain cleavage of cortisol resulting in androgen gut metabolites. These studies showed further evidence that the gut microbiota have the innate ability to monitor the host endocrine stress molecule, cortisol, and influence the metabolic fate of these endocrine hormones. 21 23-25 26,27 In addition, it has been increasingly demonstrated that gut microbiota have additional and significant neuroendocrine roles in the GBA through secondary metabolism.²2 For example, the metabolic fate in the gut of ingested tryptophan, Trp, an essential amino acid, results in indoles, serotonin (5- hydroxytryptamine, 5-HT), tryptamines, melatonin, and kynurenine. All of these metabolites have a profound effect on host mood, anxiety, sleep, and/or stress. Trp metabolism is accomplished primarily through the kynurenine pathway (95%) resulting in nicotinamide dinucleotide (NAD +) production, while only minor amounts (1%) are converted to 5-HT. Dysregulation of the kynurenine pathway can lead to aging, various mental and neurodegenerative disorders, and chronic fatigue syndrome." Whereas 5-HT has been researched for more the 70 years, its intimate role in gut physiology is just recently being understood. 28 Cortisol stimulates 5-HT reuptake in peripheral circulation through Serotonin-selective Re-uptake Transporters (SERT) further affecting 5-HT levels.30 Ninety percent of 5-HT production occurs in intestinal enterochromaffin cells (EC), and 5-HT synthesis can be stimulated through 5-HTR4 activation by the trace amine tryptamine, wherein the gut-produced 5-HT is absorbed, stored, and distributed by platelets.³ Five of the seven known classes of serotonergic receptors (5-HT1 thru 5- HT7) are expressed throughout the gut. Activation of 5-HT2a receptor by intestinal 5-HT results in a block of TNFa and subsequent increase in SERT.³ The 5-HT is distributed by platelets throughout the circulation and plays an important role in hemostasis, peristaltic reflex, and gut physiology.³5 While 5- HT does not have the ability to cross the blood brain barrier, Trp does have the ability to cross and therefore directly contributes to central and peripheral 5-HT and melatonin levels. 35,36 Sequestering of Trp through microbial secondary metabolism in the gut leads to decreased Trp in the brain and can greatly affect its bioavailability for neuroendocrine roles. Corynebacterium spp., Streptococcus spp., and Escherichia coli have been shown to synthesize 5-HT in culture. Williams et al. evaluated bacterial tryptophan decarboxylase activity (a rare functionality for gut bacteria). In fact, there is an approximately 10% prevalence in the population as discovered by Fishbach in 31,32 33,34 35 37 29 5164 Article pubs.acs.org/journal/abseba 38 39 analyzing Human Microbiome Project data. Allowing for the tryptophan decarboxylase activity in the gut would result in an increase in tryptamine and subsequent stimulation of 5-HT production from EC cells, as well as biotransformations of 5- hydroxytryptophan directly to 5-HT. In the case of chronic stress, both in animal models and human studies, subjects greatly benefited through decreasing circulating cortisol levels by increasing 5-HT levels with SRIs (Serotonin Reuptake Inhibitors).36,40 41 42 In order to mitigate the debilitating effects of chronic stress, a promising transient strategy herein was conceived to restore normal physiology and function in the gut with a whole cell engineering approach. This study utilizes a well-characterized probiotic chassis that is able to withstand and colonize the harsh gastric intestinal environment. The only commercially available Gram-negative probiotic to date, E. coli Nissle 1917, EcN, was originally isolated from a World War I soldier that was able to surprisingly survive with dysentery.4¹ The EcN strain was also recently shown to increase 5-HT extracellular concentrations in an in vitro gut tissue model." An EcN smart probiotic with elements of Clostridium scindens to sense active glucocorticoids and respond by contributing the rare Trp decarboxylase activity (similar to Clostridium sporogenes) resulted in decarboxylase activity with some substrate promiscuity toward 5-hydroxytryptophan that would result in 5-HT metabolites as well as dietary tryptophan biotransforma- tion to tryptamine. In periods of chronic stress and elevated cortisol production, we predict the engineered probiotic in the gut would increase tryptamine and ultimately intestinal 5-HT production and distribution via induced promiscuous tryptophan decarboxylase activity. The intestinal and periph- erally distributed 5-HT would drive down circulating cortisol levels and likely would improve mood, anxiety, and depression. 43 Importantly, this effort models a stress-sensing and potentially neuro-modulating probiotic in in vitro systems that prove to mimic human physiology. 44 MATERIALS AND METHODS Materials. Restriction, DNA modifying enzymes, and polymerases were from New England Biolabs (Ipswich, MA, United States). Plasmid miniprep and PCR purification kits were from QIAGEN (Germantown, MD, United States). Synthetic DNA and oligonucleo- tides were obtained from Integrated DNA Technologies (IDT; Coralville, IA, United States). Unless otherwise indicated, all other chemicals and reagents were from Sigma (St. Louis, MO, United States) or Thermo Fisher Scientific (Waltham, MA, United States). 52 Bacterial Strains and Culture Conditions. Table S1 lists the E. coli strains used in this study. Figures S9-S11 display the workflow utilized for recombinant strain construction as further detailed by Yang et al. The wild type E. coli strain Nissle 1917, referenced herein as EcN (Mutaflor, DSM 6601, and serotype 06:K5:H1), was kindly supplied by Dr. A. Breedon. BW2511, JW0451-2, and JW5503-1 were obtained from the CGSC (Yale University, Dept. of MCB), E. coli NEBSalpha (cat# C2987H) strains were obtained from NEB. The E. coli strains were routinely maintained in Luria-Bertani (LB) or M9 minimal salts medium with or without 1.5% Bacto Agar (Difco Laboratories, Detroit, Mich., United States). Electrocompetent cells were prepared using Biorad's MicroPulser (BIORAD Hercules, CA) protocol for the preparation of E. coli electrocompetent cells; see Aususbel et al. and Miller and Nickoloff. 56,57 Ampicillin (50 ug/mL), apramycin (50-100 ug/uL), chloramphenicol (30 ug/mL), and kanamycin 25-50 ug/mL) were used for antibiotic selection in this study. Biotek Plate Assay. Mid log E. coli strains were used to inoculate a fresh culture in early log (OD 600 = 0.02). Glucocorticoids https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering dissolved in culture media in 14 mm Falcon tubes (cat# 14-959-11B) with normalized DMSO 0.02-0.2% as a cosolvent and samples exposed to serially diluted glucocorticoid doses or DMSO and respective antibiotic selection. A kinetic plate assay was carried out with black/clear bottom Costar plates (cat# 3601) on a Biotek Neo2 multimodal plate reader. Both the OD 600 and the fluorescent wavelength(s) were monitored. The glucocorticoid dose response was normalized by OD600 and the EC50 was calculated with a nonlinear regression model. Data analysis was performed in Graphpad Prism 9.0. Tryptophan and Tryptamine Extraction. Following induction of tryptamine production, the culture samples were centrifuged at 16 000g for 1 min and then filtered through a 0.2 um filter, then the filtrate was treated with a 2x volume of 4N sodium hydroxide at pH 11. Next, phase extraction was performed with 1:1 with ethyl acetate, followed by mixing and centrifugation at 16 000g for 1 min to allow for phase separation. The resulting aqueous phase contained tryptophan and the organic phase captured tryptamine. Samples were subsequently prepared for HPLC analysis. Cloning and Construct Assembly. The overall cloning strategy used 2-5 DNA fragments produced from either DNA synthesis (gblocks), oligos or high-fidelity polymerase generated amplicons purified though Qiagen PCR or Qiaex gel extraction protocols. Following purification, fragments were assembled with NEB Builder HiFi Gibson Assembly using a total reaction volume of 5-10 µL. 10% of the HiFi reaction was transformed into NEB5a chemical competent cells and plated on selective media. After the plates were incubated for 18-24 h at 37 °C, individual colonies were selected for evaluation Bacterial Plasmid Construction. pLysE2Cr. The E2 crimson fluorescent reporter was designed to be under the control of LysR using the synthesized lysR with its bidirectional promoter (Genbank WP_004606447.1) included in the 250 bp upstream sequence (Figure S1) all cloned into the supplied pIDT bacterial expression vector, pIDT (kan¹), resulting in plasmid pLysRE2Cr. The lysR open reading frame (ORF) was codon optimized for E. coli expression using online in silico analysis tools provided by Integrated DNA Technologies (IDT, Coralville, IA). pLysRsfGFP. This plasmid was a gift from the U.S. Army Lab (Dr. Steve Blum) and was created by swapping the ORF of the sfGFP reporter for that of the E2 crimson reporter in plasmid pLysRE2Cr. pLysRTDC. The pLysRE2Cr plasmid was used as a PCR template for a 900bp fragment of the lysR gene (C. scindens), primed with lysR- F and lysR-R and amplified with Q5 high fidelity polymerase (NEB #M0491S). The tdc-1 (Tryptophan decarboxylase-1 from Oryza sativa) gene was synthesized by IDT as a 1.5kb fragment (see the supplemental sequence list for details) and was likewise used as a PCR template and amplified with Q5 high fidelity polymerase. The lysR and tdc-1 containing fragments were assembled via an NEB Builder HiFi Assembly kit into the XhoI/NotI sites of pAME200 (this plasmid was a gift from Dr. A. Breedon, USAF RHB) resulting in plasmid pLysTDC. PUC19lysRsfGFP-Plasmid. pLysRsfGFP was used as template with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See the supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRE2Crimson Plasmid. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200bp or 1000bp of flanking tolC homology gblocks (see supplemental sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. PUC19lysRTDC Plasmids. pLysRE2Crimson was used as templates with oligos to generate a linear fragment both assembled with either 200 bp or 1000bp of flanking tolC homology gblocks (See Supplemental Sequence information for gblocks from IDT) assembled into the pUC19 BamH1/EcoR1 vector. Colony PCR. Colony PCR was performed by touching a standard 10 ul pipet tip into the center of the bacteria colony and transferred to the bottom of a 96-well PCR plate well. NEB Quick load 2X Taq PCR 5165 Article pubs.acs.org/journal/abseba master mix was added to each well on a cooling block and once the denaturation temperature of 95 °C was reached the samples were added to the thermocycler. The initial denaturation of the PCR was conducted for 2 min. NEB Quick load 2X Taq polymerase manufacture recommendations were followed. Standard PCR. Standard PCR employed either the NEB Quick load 2X Taq polymerase protocol for routine usage or the NEB HiFi Q5 polymerase protocol for cloning and assembly efforts in this study. Flow Cytometry Analysis. Following growth and glucocorticoid treatment, E. coli strains were diluted 1:40 in phosphate buffered saline (pH 7.4) with 20mM Hoechst 33342 DNA stain (Fisher Cat. No. H21492) and incubated at 37 °C for 10 min. Following Hoechst DNA staining, all cells were stored on ice until flow cytometry data acquisition. Samples were analyzed on a 5-laser BD FACSAria II cell sorter (BD Biosciences, San Jose, CA) with instrument setup performed using Cytometer Setup and Tracking Software (BD Biosciences). Hoechst was excited with a 355 nm laser and detected with a 450/50 filter. GFP was excited with a 488 nm laser and detected with a 525/50 nm filter. E2 crimson was excited with 561 nM laser and detected with a 670/30 nm filter. During acquisition, all parameters were collected in log mode. Data were collected and analyzed using FACSDiva 8.0 software (BD Biosciences). To reduce noise, the bacteria were cultured in sterile-filtered media and diluted in sterile-filtered PBS. Prior to acquisition, the background noise of the instrument was evaluated by analyzing water-only, sheath fluid-only, and PBS-only blank samples. Forward and side scatter gates were set to exclude noise and debris; the bacterial population, identified as Hoechst- positive cells, was further analyzed to identify single cells (FSC-height vs FSC-width, followed by SSC-height vs SSC-width). Positivity gates for the different fluorescent parameters were set after analyzing multiple controls (unstained/untreated culture, unstained/treated culture, stained/ untreated culture). Flow cytometry analyses are in the supplementary. 53 Gene Knockout Generation. Ato/C:CAT Knockout in E. coli Nissle 1917. A knockout strain was constructed using a traditional Red Recombination protocol by Datsenko and Wanner. The chloramphenicol acetyltransferase, CAT, gene in pKD3 was utilized as template to generate a CAT flanking 50 bp tolC homology PCR cassette (See Figure 4) using oligos C1 and C2. Electrocompetent EN1917 cells expressing the red recombinase helper plasmid, pKD46, were transformed with the resultant PCR product targeting the tolC gene. Cells recovered for 1 h and selection was performed on LB chloramphenicol plates (15 ug/mL). AtolC:Ap' Knockout in E. coli Nissle 1917. The apramycin resistance gene, Ap', in pMDIAI was utilized as a PCR template primed with oligos A1 and A2 to generate an Ap¹ PCR fragment with flanking 50 bp homology to tolC. According to a published protocol by Yang et al.,52 electrocompetent EcN cells expressing the pREDTKI plasmid were transformed with the PCR product targeting the tolC gene. Cells were recovered in 1 h and plated on LB apramycin selection plates (50 ug/mL) and individual colonies were selected for further analysis. 52 Targeted Gene Integration. Integration of donor cassettes from lysRsf GFP, lysRE2Crimson, and lysRtdc-1 into the tolC locus was accomplished according to published protocols by Yang et al.5² The intermediate strain AtolC:Ap¹ EcN was used as a recipient strain for targeted integration into the tolC locus. See Figures S11, S12, and S13 for workflow and an example of the screening and validation. Tryptamine Production. Early log (OD600 = 0.02) strains were grown in M9 media with 1% LB media and 2% glucose with 6 mM L- tryptophan and were maintained under selection of chloramphenicol 30ug/mL. Following an OD600 = 0.2, cultures were induced with the stated quantities of cortisol. Cultured samples were collected at the stated times and centrifuged at 16 0000g for 1 min to remove biomass and were subsequently prepared for chemical analysis. 1. RESULTS AND DISCUSSION 2.1. Designing a Novel Cortisol Biosensor. To design a real-time probiotic stress sensor, the bacterial chassis, E. coli https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering 45-47 Nissle 1917 was employed, due to the probiotic's ability to survive the challenging gastrointestinal environment and its reported use as a framework for synthetic biology. The use of a whole cell approach affords the ability to utilize a transcription factor responsive to cortisol that can activate a downstream transcriptional response (i.e., reporter gene or stress-reducing factor). Published RNA-seq data for C. scindens ATCC 35704 demonstrated an effect for its neighboring regulon upon exposure to cortisol (Figure S1).³ LysR-type transcriptional regulators (LTTRs) are the largest prokaryotic class of transcription regulators. In C. scindens, LysR regulates its own transcription as well as its native divergently transcribed gene product. When LysR disassociates from its own promoter, lysR transcription is active, whereas when bound to the coinducer (i.e., cortisol), promoter activation results in transcription of the divergently located gene (Figure 1). Based on this design, the lysR nucleotide sequence was LysR lysR LysR LysR I Co LysR Co lysR Figure 1. Schematic of the LysR regulation of transcription in Clostridium sp. placed under native control elements for expression in E. coli K-12 and EcN strains and evaluated activation of various LysR responsive reporter genes upon cortisol induction. 2.2. Employing a Novel Cortisol Biological Recog- nition Element, BRE. In Clostridium sp. Ridlon et al. demonstrated that the lysR and its divergently transcribed gene product is transcriptionally active in the presence of cortisol, based on RNA-seq data analysis.³ Preliminary analysis was performed in order to determine if the lysR operon had the ability to function as a glucocorticoid transcriptional driven sensor, by employing the Clostridium scindens lysR native control elements driving the bidirectional expression of the E2 Crimson fluorescent reporter protein. The E2 Crimson fluorescent protein has an excitation maximum at 611 nm and emission maximum at 646 nm, is nontoxic to bacteria and performs well with live animal imaging.* The pLysE2Cr plasmid bears the kanamycin resistance gene and the pMB1 origin of replication. Chemical transformed E. coli K-12 and EcN bearing the plasmid pLysE2Cr following mid log growth were evaluated for their dose response to cortisol by measuring fluorescence with a plate reader and flow cytometry. In Figure 2, the schematic demonstrates that the LysR negatively regulates its own transcription (gold) and positively regulates the divergent reporter gene (green) with putative LysR responsive promoters (blue). Exposing the EcN probiotic bearing the pLysE2Cr to varying doses of cortisol (coinducer in Figure 2) at mid log demonstrated a definitive dose response 48 5166 pubs.acs.org/journal/abseba curve with an EC50 of 158 µM and is supra-physiological, whereas the normal human physiological cortisol concen- trations in plasma observe a diurnal variation and range between 80 and 700 nM, and the water solubility of cortisol is 772 μM. Multiparametric flow cytometry analysis was used in this study, since it affords reproducible and accurate functioning at the single cell level and fluorescent reporter proteins afford accurate representations of isogenic cultures. In the EcN wild type strain bearing the pLysE2Cr plasmid, E2 Crimson fluorescent protein expression was evaluated via flow cytometry following incubation with varying concentrations of cortisol. In Figure 2, the top panel/density plots, show a dose- dependent shift of the cell population into the E2 Crimson positive gate, indicating an increased percentage of cells expressing E2 Crimson in response to cortisol. In Figure 2, the bottom panel histograms show a dose-dependent increase in the population's median fluorescence intensity with increasing cortisol concentration. Evaluation of additional reporters such as sfGFP and iLuX (Figure S3) gave similar results. 2.3. Cortisol BRE Selectivity for a-THF, an Active Cortisol Metabolite. In an effort to determine the glucocorticoid specificity of the sensor, varying concentrations of cortisol and its associated metabolites were evaluated on early growth phase EcN bearing the engineered pLysE2Cr plasmid. Both cortisol and its active metabolite 5-alpha- tetrahydrocortisol (THF) were able to transactivate LysR and drive E2Cr reporter expression in similar percentage of the cell population (73% and 59%, respectively) as demonstrated in the density and histogram plot in Figure 3B. Furthermore, cortisol and THF demonstrated robust dose responses with similar EC50 values (213 µM and 150 μM respectively, Figure 3C). The inactive cortisol metabolite of cortisone, 5-beta- tetrahydrocortisone (THE), was unable to initiate the E2Cr reporter in a dose dependent fashion (Figure S4), further indicating the selectivity of the pLysE2Cr sensor for active glucocorticoids. Article 49 The lumen of the gut is a harsh environment with additional cortisol analogs present, such as bile salts. Bile salts are amphipathic steroid molecules found exclusively in the G.I. tract and bile salts are critical for the absorption of dietary lipophilic foodstuffs in the lumen of the small intestine because of their capacity to form micelles spontaneously in a concentration dependent manner. Deoxycholate (DOC), a secondary bile salt (typically present at 20 µM to 100 µM in the small intestines) was investigated to see if DOC would elicit a response with the LysR sensor. DOC afforded a dose response (Figure S5) with an EC50 of 1.5 mM, although these supra-physiological (>1 mM) quantities of DOC proved deleterious to ECN growth (Figure S5).5⁰ 2.4. Targeted Ablation of Glucocorticoid Efflux in E. coli Nissle 1917 to Improve Glucocorticoid Sensor Sensitivity. Although the LysR sensor is able to selectively recognize cortisol (Figure 3), the limit of cortisol detection by the LysR sensor in the wild type EcN host is not physiological relevant (EC50 = 158 µM) and requires further improvements. Available acrAB and tolC mutants (Keio collection mutants JW0451 and JW5503) generated in the wild type parental strain E. coli BW25113 were tested for cortisol sensitivity. Both acrAB and tolC mutants demonstrated a 2-log improvement in cortisol sensitivity (Figures S6 and S7). Following this, the tolC locus was ablated in EcN by directing the chloramphenicol acetyltransferase gene (CAT) gene to the tolC locus using Red homologous recombination (data not shown). PCR- https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175 ACS Biomaterials Science & Engineering A. B. C. 530/30 Blue-A 10² 10³ 104 D. LysR family TF Regulator -57 plysE2 Crimson lysR ΟμΜ Singlets 0 10 -81 %E2Cr+, Normalized Response 10 0 10 10 105 E2Crimson 670/30 YG-A Singlets 0% E2Crimson+ E2Crimson MdFI 10⁹ 10 10° E2Crimson 670/30 YG-A 100 50 10⁰ 530/30 Blue-A -65 -65 10² 62.5 μΜ Singlets 0 10² 10 Singlets pubs.acs.org/journal/abseba 10 10 E2Crimson 670/30 YG-A verified AtolC:CAT ECN mutants (data not shown) were transformed with the pLysE2Cr plasmid (Figure 4A) and 10 0.6% E2Crimson MdFI 0 10² 10ª 105 E2Crimson 670/30 YG-A E2Crimson+ 5167 reporter/response E2 Crimson 530/30 Blue-A 10² 10³ 104 105 106 Count 500 1.000 1.500 2,000 46 pMB1 ori 0 125 μM Singlets TIMME TIITING. ITIINI 10 10ª 10$ E2Crimson 670/30 YG-A 28% E2Crimson+ Singlets E2Crimson MdFI 10⁰ E2Crimson 670/30 YG-A 10² 10ª 10⁰ 158μM = EC50 104 [Cortisol nM] Figure 2. A novel cortisol responsive transcription factor drives reporter expression. (A) Schematic of the LysR operational unit, where LysR negatively regulates its own promoter and lysR (gold) and drives the expression of the reporter or response gene (green). (B) Plasmid map of pLysE2 Crimson. (C) Flow cytometric analysis of pLysE2 Crimson in EcN with 0, 62.5, and 125 uM of cortisol. Top row, density plots: the gates are indicative of positive E2 Crimson expression. Bottom row, histograms: the interval gates measure the MdFI, median fluorescence intensity. (D) Cortisol dose response curve (EC50 = 158 uM) of pLys E2Crimson in wild type EcN. Article evaluated in mid log cultures for a cortisol dose response (Figure 4C). The AtolC:CAT mutation in EcN enhanced https://doi.org/10.1021/acsbiomaterials.2c01300 ACS Biomater. Sci. Eng. 2023, 9, 5163-5175/nSee Answer
Q9: Review Films for Wound Healing Fabricated Using a Solvent Casting Technique pharmaceutics Fabiola V. Borbolla-Jiménez ¹,2D, Sheila I. Peña-Corona Emiliano Pineda-Pérez ¹,2D, Alejandra Romero-Montero María Luisa Del Prado-Audelo ², Sergio Alberto Bernal-Chávez 4, Jonathan J. Magaña ¹,²,* and Gerardo Leyva-Gómez ³,*D I Citation: Borbolla-Jiménez, F.V.; Peña-Corona, S.I.; Farah, S.J.; Jiménez-Valdés, M.T.; Pineda-Pérez, E.; Romero-Montero, A.; Del Prado-Audelo, M.L.; Bernal-Chávez, S.A.; Magaña, J.J.; Leyva-Gómez, G. Films for Wound Healing Fabricated Using a Solvent Casting Technique. Pharmaceutics 2023, 15, 1914. https://doi.org/10.3390/ pharmaceutics15071914 check for updates Academic Editors: Giyoong Tae, Martin Federico Desimone and Gorka Orive CC Received: 18 May 2023 Revised: 10 June 2023 Accepted: 27 June 2023 Published: 9 July 2023 4.0/). BY 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/ 1 2 3 4 * 3 D, Sonia J. Farah ¹,2, María Teresa Jiménez-Valdés ¹,2, 3 Laboratorio de Medicina Genómica, Departamento de Genómica, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, Ciudad de México 14389, Mexico; fvbj@hotmail.com (F.V.B.-J.); soniafarah1d@gmail.com (S.J.F.); teresajimenez00@gmail.com (M.T.J.-V.); emilianopineda_perez@hotmail.com (E.P.-P.) Tecnologico de Monterrey, Campus Ciudad de México, Ciudad de México 14380, Mexico; luisa.delprado@tec.mx Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico; sheilairaispc@gmail.com (S.I.P.-C.); alejandra.romero.montero@outlook.com (A.R.-M.) MDPI Departamento de Ciencias Químico-Biológicas, Universidad de las Américas Puebla, Ex-Hda. de Sta. Catarina Mártir, Cholula 72820, Puebla, Mexico; q901108@hotmail.com Correspondence: magana.jj@tec.mx (J.J.M.); leyva@quimica.unam.mx (G.L.-G.) Abstract: Wound healing is a complex process that involves restoring the structure of damaged tissues through four phases: hemostasis, inflammation, proliferation, and remodeling. Wound dressings are the most common treatment used to cover wounds, reduce infection risk and the loss of physiological fluids, and enhance wound healing. Despite there being several types of wound dressings based on different materials and fabricated through various techniques, polymeric films have been widely employed due to their biocompatibility and low immunogenicity. Furthermore, they are non-invasive, easy to apply, allow gas exchange, and can be transparent. Among different methods for designing polymeric films, solvent casting represents a reliable, preferable, and highly used technique due to its easygoing and relatively low-cost procedure compared to sophisticated methods such as spin coating, microfluidic spinning, or 3D printing. Therefore, this review focuses on the polymeric dressings obtained using this technique, emphasizing the critical manufacturing factors related to pharmaceuticals, specifically discussing the formulation variables necessary to create wound dressings that demonstrate effective performance. Keywords: skin; wound; wound healing; wound dressings; polymers; films; solvent casting 1. Introduction A wound is a disruption of the continuity of body tissue caused by physical or chemical damage. Usually, a wound is disinfected using a typical medical procedure, and antibiotic treatment is initiated to start the healing process [1]. However, a possible lack of response to antibiotics or an uncontrolled inflammatory phase can trigger the generation of chronic or infected wounds, which represents a clinical challenge [2]. In this sense, developing minimally-invasive smart dressings that integrate drug release with different therapeutic targets (such as antibiotics, anti-inflammatory agents, and analgesics) represents a desirable alternative [3,4]. Based on understanding the physiological process of wound healing, the ideal dress- ing should be biocompatible, acting as a physical barrier against microorganisms while allowing gas permeation to keep the wound hydrated and remove excess exudate [5,6]. Additionally, desirable properties include good mechanical strength and flexibility. Non- toxicity, biocompatibility, and biodegradability are also important criteria for materials used Pharmaceutics 2023, 15, 1914. https://doi.org/10.3390/pharmaceutics15071914 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2023, 15, 1914 in dressings [7]. Hydrogels, polymer films, foams, gauzes, and hydrocolloids are among the most extensively studied dressings, depending on the wound type and therapeutic needs [8,9]. Films serve as convenient physical barriers to bacteria, maintain gas permeability, and enable in situ drug release. Furthermore, their flexibility can be tailored to accommodate individual morphology [10]. Consequently, research focuses on utilizing film dressings as drug carriers to control infections and inflammatory processes [11]. By providing a moist environment, removing wound exudates, and accelerating cellular and tissue regeneration, dressings can maintain optimal conditions for wound repair [12]. However, it is important to acknowledge the limitations of these products. For example, they encounter difficulties in simultaneous application to both external and internal wounds. Additionally, their application can exert external pressure, leading to secondary injuries [13]. This study aims to analyze the solvent casting procedure, the most widely used method for film production. Solvent casting is preferred over other methods, such as salt leaching, spin coating, microfluidic spinning, and 3D printing, due to its cost-effectiveness, simplicity, practicality, and ability to generate robust films with appropriate mechanical properties and homogeneity. However, the properties of materials obtained through solvent casting can vary significantly between production batches, influenced by environmental conditions, which can also slow the production process. Moreover, maintaining sterility throughout all manufacturing steps poses a challenge and can result in batch contamination. Thus, this study comprehensively reviews the objectives of developing polymer films and highlights the key considerations to ensure optimal final properties during the solvent casting process. Despite the method's considerable potential for industrial scaling and clinical application, there is a noticeable lack of standardized commercial products [7]. Therefore, this review identifies areas for improvement and underscores the main advan- tages of utilizing this type of dressing in wound treatment. 2. Wound Healing The wound healing process is a natural physiological reaction to tissue injury that consists of four highly integrated phases: hemostasis, inflammation, proliferation, and remodeling (Figure 1). These phases must occur in a specific sequence, at a specific time, and for a particular duration, in order that the physiologically involved functions fulfill their expected role [14]. Otherwise, this leads to improper or impaired tissue repair [15]. Epidermis Dermis Fibrin clot Erythrocyte- Blood_ vessel 1) Hemostasis Proliferating fibroblasts Epithelial cells monolayer New blood vessels -Injury 3) Proliferation Dobago -Platelet -Eschar Type III Collagen fibers Granulation tissue (2) Inflammation Necrotic tissue or bacterias Macrophage- Neutrophil- Monocyte 2 of 27 4) Remodeling Scar tissue Fibroblast. Type I Collagen fibers Figure 1. Four significant phases represent the wound healing process: (1) hemostasis, the formation of a platelet seal that prevents blood loss and a fibrin clot; (2) inflammation, where neutrophils and macrophages remove debris and prevent infection; (3) proliferation, where blood vessels reform Pharmaceutics 2023, 15, 1914 3 of 27 through angiogenesis, and fibroblasts replace the fibrin clot with granulation tissue; (4) remodeling, where the matrix is remodeled replacing type III collagen with type I, maturing to a scar. 2.1. Hemostasis The hemostasis phase begins minutes to hours after an injury through a cascade of serine protease activation, resulting in platelet activation. This activation also facili- tates the release of growth factors, such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), and TGF-α (transforming growth factor x), as well as immune mediators, contributing to the transition into the inflammatory phase [16]. PDGF and TGF-ß (transforming growth factor ß) recruit neutrophils and monocytes to initiate the inflammatory response [17]. Furthermore, this leads to the formation of a fibrin clot, referred to as an eschar, which acts as a plug for the wound, preventing blood loss, providing a scaffold for incoming immune cells, serving as a reservoir for cytokines and growth factors during the early stages of repair, and offering protection against bacterial invasion [15,16,18]. The extrinsic clotting cascade is initiated upon tissue damage and blood leakage, releasing molecules such as serotonin that induce localized vasoconstriction [19]. Sub- sequently, platelets aggregate and become activated upon contact with subendothelial collagen, forming a hemostatic plug. This plug mitigates hemorrhage and serves as a provisional matrix for cell migration by releasing scaffold proteins such as fibronectin, vitronectin, and thrombospondins, facilitating the migration of keratinocytes, immune cells, and fibroblasts [16,20]. Once a fibrin clot is formed, the coagulation process is switched off to prevent excessive thrombosis [17]. 2.2. Inflammation The inflammatory phase, which overlaps with hemostasis, occurs during the first 72 h [16]. During this phase, both humoral and cellular inflammatory responses are activated to establish an immune barrier against invading microorganisms [21]. Neu- trophils and monocytes infiltrate the wound bed to prevent further damage and eliminate pathogenic organisms and foreign debris [16,22]. The inflammation phase can be divided into early and late inflammatory phases. In the early phase, the complement cascade is activated, leading to the infiltration of neutrophils into the wound. Their primary role is to phagocytose bacteria, foreign particles, and damaged tissue [21,23]. Various chemoattractive agents attract neutrophils to the wound site, including TGF-ß, complement components such as C3a and C5a, and formyl- methionyl peptides produced by bacteria and platelet products [16,19]. During their activity, neutrophils release proteolytic enzymes, oxygen-derived free radical species, and inflammatory mediators such as TNF-α and interleukin (IL)-1 [17,21]. Once their role is fulfilled, neutrophils undergo apoptosis and are cleared from the wound typically within 2-3 days, making way for the influx of monocytes [18]. Monocytes, stimulated by cytokines, chemokines, growth factors, and soluble frag- ments of the extracellular matrix (ECM), differentiate into activated macrophages. These macrophages patrol the wound area, ingesting and killing bacteria and removing devi- talized tissue through the action of secreted matrix metalloproteinase and elastase [15]. Additionally, macrophages play a crucial role in transitioning to the proliferative phase by releasing various growth factors and cytokines, including PDGF, TGF-α, TGF-ß, insulin-like growth factor-1 (IGF-1), fibroblast growth factor (FGF), tumor necrosis factor-a (TNF-α), IL-1, and IL-6. These soluble mediators promote cell proliferation and the synthesis of ECM molecules, and activate fibroblasts for subsequent phases [19,24]. A decrease in macrophage presence within the wound indicates the inflammatory phase's conclusion and the proliferative phase's initiation [15]. Pharmaceutics 2023, 15, 1914 4 of 27 2.3. Proliferation The proliferative phase of wound healing involves several interconnected processes that restore tissue structure and function. It begins on the third day after the injury and lasts approximately two weeks [21]. One of the critical aspects of this phase is the replacement of the provisional fibrin matrix with a new matrix composed of collagen fibers, fibronectins, and proteoglycans, which are synthesized by fibroblasts. During this phase, angiogenesis, the generation of granulation tissue, collagen deposi- tion, re-epithelialization, and wound contraction occur [16,25]. Angiogenesis is stimulated by local conditions such as low oxygen tension, low pH, and high lactate levels. It involves the migration and proliferation of endothelial cells to form new blood vessels, which is cru- cial for tissue viability [17]. Macrophages are vital in promoting angiogenesis by producing VEGF [16]. Granulation tissue formation occurs concurrently with angiogenesis and primarily comprises type III collagen, fibroblasts, and new vessels. Fibroblasts are the main cells involved in granulation tissue formation. Their proliferation, and synthesis of extracellular matrix components, contributes to tissue restoration [21]. Additionally, the interaction between fibroblasts and keratinocytes plays a significant role in re-epithelialization. Factors like EGF, KGF, and TGF-α, produced by platelets, keratinocytes, and anti-inflammatory macrophages, promote keratinocyte migration and proliferation [25-27]. The process is further facilitated by the production of fibronectin, tenascin C, and laminin 332 by keratinocytes [16]. As re-epithelialization progresses, the stratified layers of the epidermis are re-established, and the maturation of the epidermis begins to restore its barrier function. TGF-ß can accelerate this maturation process [17]. The proliferation and differentiation of keratinocytes are essential for the reestablishment of a functional epidermis [25,27]. 2.4. Remodeling The remodeling phase, which occurs several weeks after the initial wound, marks the transition from granulation tissue to scar formation. This phase may last up to 1-2 years [21]. During this phase, angiogenesis slows down, and type III collagen in the granulation tissue is replaced by stronger type I collagen [16]. Myofibroblasts play a crucial role in driving the remodeling phase. They originate from fibroblasts and develop in response to mechanical tension and TGF-ß signaling. Myofibroblasts are responsible for wound contraction, and their expression of smooth muscle actin (SMA) generates the contractile force exhibited by these cells [28,29]. It is worth noting that myofibroblasts are considered terminally differentiated and undergo apoptosis once the remodeling process is complete [16]. During the remodeling phase, the granulation tissue matures into a scar, and the tensile strength of the tissue increases. This maturation is characterized by a reduction in the number of capillaries, an aggregation into larger vessels, and a decrease in the number of glycosaminoglycans. The cell density and metabolic activity in the granulation tissue also increase during this maturation process. The collagen type and organization changes further enhance the tissue's tensile strength [17]. However, the tensile strength of the healed tissue never fully reaches its original strength. A newly epithelialized wound typically has approximately 25% of the tensile strength of normal tissue, and it may take several months for the tensile strength to increase to a maximum of 80% of normal tissue [30]. The enhancement of tissue tensile strength primarily occurs through reorganizing collagen fibers, initially deposited randomly during the granulation phase. After, the enzyme lysyl oxidase, secreted by fibroblasts into the extracellular matrix (ECM), facilitates the increased covalent cross-linking of collagen molecules [17]. Pharmaceutics 2023, 15, 1914 Type of Polymer Natural Synthetic 3. Polymeric Films for Wound Healing Polymeric films (PFs) as dressings for wound healing were first vastly introduced dur- ing the Second World War as a response to the demand for advancements in medicine [31]. Bloom et al. [17] were the first to document semipermeable films in 1945 using cellophane to treat burnt prisoners of war in Italy; they reported “gratifying results” with complete healing nine days after dressing application [32]. Nowadays, PFs are commonly utilized in the medical field for healing as physical barriers for wounds which help to prevent inflammation, control the environment of the wound, and accelerate healing [33]. PFs have gained popularity due to their non-invasive nature, ease of application, biocompatibility, and the potential inclusion of antimicrobial treatments. Moreover, PFs offer flexibility, adherence, gas exchange capabilities, and trans- parency [34]. The material's flexibility enables the film to conform to complex shapes while facilitating gas exchange, which has been proven to promote healing [34]. Additionally, its transparency allows for the close monitoring of the wounded area without removing the film, thereby reducing trauma during dressing changes, minimizing exposure to bacteria, and lowering the risk of infection by up to one week [34,35]. PFs are recommended for treating partial-thickness wounds, minor burns, lacerations, and certain low-exudate ulcers such as ischemic ulcers, diabetic ulcers, venous ulcers, and pressure ulcers [34]. However, it is important to note that due to the occlusive nature of the material, films should not be used on wounds that require significant fluid absorption, as excess exudate can potentially lead to peri-wound maceration. Nevertheless, maintaining a certain level of moisture can be beneficial for the healing process by promoting keratinocyte migration to the affected area [34,36]. The performance of PFs depends on the chemical composition and the type of wound. Polymers used to formulate PFs can be divided into natural, synthetic, and blended. Therefore, the PFs will possess different qualities depending on the type of polymers employed to form the film dressing. 3.1. Natural Polymeric Films Natural polymers, also called biopolymers, offer advantages such as biocompatibility, biodegradability—at present a sought-after quality-healing properties, inertness, and adhesiveness [37]. For this reason, generally, natural polymers are preferred over synthetic ones [37]. However, they are prone to microorganism contamination and lack quality mechanical properties to form an optimal polymeric film. Some examples of natural polymers used for elaborating PFs are chitosan, hyaluronic acid, starch, silk fibroin, sericin, keratin, sodium alginate, gelatin, collagen, zein, and konjac glucomannan, among others (Table 1) [36]. Chitosan is one of the most exploited and abundant natural polymers in wound dressings, with antimicrobial and film-forming properties [38,39]. Moreover, due to their innocuousness, polymers like chitosan, cellulose, gellan gum, alginates, and starches can be used for oral cavity films [40] (Figure 2). Table 1. Types of polymers used in polymeric film casting. Examples Chitosan, hyaluronic acid, starch, silk fibroin, sericin, keratin, sodium alginate, gelatin, collagen, zein, cellulose, and konjac glucomannan Polyvinyl alcohol, polyacrylic acid, polycaprolactone, polyethylene glycol, polyvinylpyrrolidone, polylactic acid, and polydimethylsiloxane. 5 of 27 Properties Biocompatibility, biodegradability, high disponibility, healing properties, permeability, inertness, and bioadhesiveness Resistance, flexibility, structure, high degree of polymerization, thermo-responsiveness, hydrophilicity, and occlusivity Ref. [36,37] [36]See Answer

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