The growing demand for more sustainable, alternative processes leading to production of plant-derived preparations imposes the use of plants waste generated mainly by agri-food and pharmaceutical industries. These mostly unexploited but large quantities of plants waste also increase the interest in developing alternative approaches for sustainable production of therapeutic molecules. In order to reduce the amount of plant waste by further processing, different novel extraction techniques can be applied. Fruits and their industrial by-products are rich sources of different classes of compounds with therapeutic properties. The processed fruits waste can be reused and lead to novel pharmaceuticals, food supplements or functional foods. This review intends to briefly summarize recent aspects regarding the production of different active compounds from fruit by-products, and their therapeutic properties. The potential use of fruits by-products in different industries will be also discussed. According to the United Nation’s Food and Agriculture Organization, the production of different fruits around the world exceeded one billion tones in 2017, which, depending on geographical areas, consumption and growing traditions, inevitably leads to the generation of large amounts of by-products and waste . Only EuThe most common fruits consumed worldwide are apples, grapes, and exotic fruits typical for the cultivation region. Apples are the most popular fruits that provide production of juice and cider production in over three million tons in Europe  and abouAccording to international statistics, grapes are world’s largest fruit crop, with an annual production of over 75 million of tons worldwide . In thisOther fruits with a large production are the exotic fruits like coffee, macadamia, pineapple, papaya, and mango. They are rich in vitamins, carotenoids, phenolic compounds and dietary fiber distributed in different parts of the fruits  .In this context, our short review intends to present a critical opinion on aspects regarding the production of active compounds from different fruits by-products, their therapeutic properties and the potential use of fruit by-products in different industries, being a link for researchers from different sciences: chemistry, pharmacology and medicine.Scientific development registered in the last decades in the field of extraction of biologically active molecules enables the valorization and consequently utilization of fruit processing by-products. Thousands of valuable molecules generated from fruits waste can be used in food, cosmetic, or pharma industry . In ordestate-of-the-art techniques. However, in this case, equipment needed and optimization parameters are important issues for obtaining active compounds from by-products. The most suitable and frequently used for isolation of polyphenolic compounds from fruits by-products are extraction techniques involving pressure, microwave and ultrasounds. In order to increase the yield of bioactive compounds and decrease amount of solvents used, and energy consumption, extraction techniques usually involve some pre-treatment and post-treatment processes. Pre-treatment processes are typically applied to remove lignin, reduce cellulose crystallinity and increase cell porosity , High Voltage Electrical Discharges (HVED) or Pulsed Ohmic Heating (POH) have been investigated, mostly for the recovery of phenolic compounds related to valorization of fruits wastes and by-products. PEF enabled to obtain high content of polyphenols from vine shoots  . HVED prThe important challenge in the recovery of phenolic compounds from fruits waste is the choice of the solvent used, which must meet the REACH characteristics  . Bio-basThe economic feasibility of the extraction method depends on yield of recovery of active compounds. Due to the diversity of constituents present in fruit waste, a fractionation strategy would improve the efficiency and cost of the processes. When the process involves the recovery of several compounds from fruits by-products, a major concern to consider is the order of extraction. Due to mechanic processes like pressing, phenolic compounds may bind to cell wall, leading to decreased extraction yield. This interaction reduces also the extractability of pectins. Therefore, in any given process designed to obtain more than phenolic compounds, these should be extracted first. Moreover, the fact that the extraction process is time and solvent consuming, the order of extraction of the compounds can be the bottleneck of the entire biorefinery process .in vitro (in vitro (Prunus tomentosa seed waste inhibited α-glucosidase at the concentration level comparable to positive control acarbose and exhibited significant protective effect against H2O2-induced HepG2 cells damage (in vitro (Therapeutic properties of bioactive compounds derived from fruits are widely described in the scientific literature. A number of reviews summarized anticancer, antidiabetic, antihypertensive, anti-inflammatory, antimicrobial, antioxidant, immunomodulatory, or neuroprotective activity of plant secondary metabolites extracted from fruits, underlining their importance in the human diet . Scientiin vitro . Orange in vitro . Ellagitin vitro . Pomegrain vitro . Aromatis damage . Catechiin vitro , while ain vitro . Also, pin vitro .in vitro studies have to be confirmed not only in animal models, but also in clinical trials. Anyhow, standardized extracts or isolated active compounds incorporated into dietary supplements or functional cosmetics can also be a stream for fruits waste management. The re-utilization of food by-products is the easy way for sustainable production of active compounds and this idea gained recently a lot of interest. With the application of efficient extraction techniques fruits by-products can become a cheap source of active compounds.High biological potential of redundant fruit biomass can be utilized by pharmaceutical industry. However, processing of fruits waste for possible medicinal applications requires strict standardization of extracts or determination of purity of isolated compounds. What is more, before ingredients of fruits waste can be applied in a form of drugs, preliminary Large amounts of wastes and by-products are produced at industrial levels, most of them containing highly valuable bioactive substances, which can be further used for different applications . Hence, Several applications of compounds recovered from different industries are well known. Compounds recovered from fruits wastes find further applications as additives in food products to preserve and enhance quality, to prevent food oxidation and to inhibit the growth of pathogenic microorganisms . Also, aAspergillus versicolor (Aspergillus flavus (Due to rich composition in sucrose, inulin, maltose, glucose, fructose, starch, galactose, dextrose or lactose, fruits waste can be used as substrate for production of oligosaccharides or microbial polymers . Conventrsicolor  or Aspers flavus . The agrs flavus . Fruits s flavus  or bioets flavus . Most ins flavus .Fruits by-products can be considered a rich source for the recovery and production of multiple co-products in an integrated biorefinery model where green methods can be combined in order to obtain added-value products. The method used for the recovery of active constituents and the selection of the appropriate solvent have a great influence on the extractability of the functional compounds, no unique extraction procedure being suitable for all samples and matrixes.Valuable compounds can be directly formulated as nutraceuticals for their proven health benefits or can be used as raw materials for other industries. The concept of recovery of different compounds from these wastes opens new routes for the development of “green” industries, which have a tremendous potential, especially where the availability of fruits waste is abundant. The emphasis on frontier sciences has already attracted interest in developing and optimizing new ecological methods for the efficient use of the biomass, for closing the chain by returning nutrients and organic matter to the soil, when all other useful products have been recovered.RF, IF, and ES contributed to data collection and analysis, manuscript design and preparation. AO and JX revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fractional exhaled nitric oxide (FeNO) is a well-known and widely accepted biomarker of airways inflammation that can be useful in the therapeutic management, and adherence to inhalation therapy control, in asthmatic patients. However, the multiple-flows assessment of FeNO can provide a reliable measurement of bronchial and alveolar production of NO, supporting its potential value as biomarker also in peripheral lung diseases, such as interstitial lung diseases (ILD). In this review, we first discuss the role of NO in the pathobiology of lung fibrosis and the technique currently approved for the measurement of maximum bronchial flux of NO (J’awNO) and alveolar concentration of NO (CaNO). We systematically report the published evidence regarding extended FeNO analysis in the management of patients with different ILDs, focusing on its potential role in differential diagnosis, prognostic evaluation and severity assessment of disease. The few available data concerning extended FeNO analysis, and the most common comorbidities of ILD, are explored too. In conclusion, multiple-flows FeNO analysis, and CaNO in particular, appears to be a promising tool to be implemented in the diagnostic and prognostic pathways of patients affected with ILDs. The term fractional exhaled nitric oxide (FeNO) indicates a quantitative analysis of a gas, nitric oxide, in exhaled air. The presence of nitric oxide in exhaled air was first demonstrated through chemiluminescence analysis and mass spectrometry by Gustafsson in 1991 ,3.Thus, the possibility to measure a biomarker of airway inflammation with a noninvasive, reproducible and economic technique led to the development of exhaled NO analyzers able to quantify the NO burden in the airways. In the last two decades, FeNO emerged as one of the most important biomarkers in the management of bronchial asthma, and the procedure for its measurement was first standardized in 2005 by ATS and ERS society . ConcernHere, we perform a systematic review of literature in order to report all the available evidence concerning the rationale and the potential usefulness of extended FeNO analysis in the clinical management of ILDs.We perform a systematic search of PUBMED scientific papers in the English language. We used the keywords interstitial lung diseases, extended nitric oxide analysis, multiple-flows exhaled nitric oxide, and alveolar nitric oxide to discriminate potentially relevant studies. Reference lists of selected studies were also evaluated to obtain additional sources of evidence. We excluded conference papers, editorials, short letters and commentaries. The role of NO in the pathogenesis and or pathophysiology of ILDs is not fully understood although oxidative stress is well recognized as an essential component for the development of lung fibrosis, particularly in idiopathic pulmonary fibrosis (IPF) ,11. The Substantial evidence suggesting the important role of NO in fibrogenesis has been published on systemic sclerosis-associated ILD (SSC-ILD). Despite its protective effect in terms of vasodilation, an overproduction of NO was demonstrated in affected tissues (skin and lung in particular), suggesting a dual activity of NO in different phases of disease . This diStill, the role of NO in lung fibrogenesis remains controversial. A recent paper by Noguchi et al. showed that triple knockout of the three isoforms of NOS  led to a significant deterioration of lung fibrosis that could be reverted with supplemental NO , suggestTherefore, more studies are needed to understand how the imbalance among different NOS can impact on the development of lung fibrosis, focusing on the potential protective/detrimental role of different patterns of enzymatic expression and/or specific cellular sources of NO.Many mathematical models have been proposed in the last two decades to properly discriminate and measure NO from different pulmonary districts ,29,30. AILDs are a very heterogeneous group including more than two hundred different histopathological disease entities. The majority of ILDs are rare, and the diagnosis is often a real challenge, requiring a multidisciplinary discussion and invasive diagnostic approaches in a relevant percentage of cases. Thus, scientific research has focused on the discovery and validation of biomarkers that could help clinicians in the diagnostic pathway to avoid more invasive and expensive exams. Among these biomarkers, extended FeNO analysis can be considered a very interesting tool because it is reproducible, easy to obtain, pain-free and relatively cheap. Therefore, many studies have been published to investigate its potential role in the clinical management of different ILDs. In SSC-ILD represents the most common among the subgroup of connective tissue disease-related ILDs (CTD-ILD). The early recognition and treatment of SSC-ILD is crucial in the management of these patients, as the development of lung fibrosis is a crucial prognostic factor because it constitutes the most common cause of death of SSC patients ,57. MoreFeNO has been studied in SSC-ILD patients since 1997, reporting conflicting results due to the lack of standardization of sampling methods and the inclusion in study populations of patients with and without different lung comorbidities  ,60,61,62The current limitations of extended FeNO analysis in SSC-ILD are that no specific studies have compared CaNO with other prognostic biomarkers in SSC-ILD. As well, further studies are needed to properly assess the value of this biomarker in estimating survival in these patients.Data concerning the potential role of multiple-flow analysis of FeNO in IPF has been growing in the last years. Although IPF is the most common and lethal among diffuse ILDs, diagnosis still represents a relevant challenge for clinicians, as well as the prediction of clinical course, that remains unpredictable ,63. A grThus, the possibility to obtain a reliable and noninvasive biomarker from exhaled breath, with a theoretically lower risk of sample contamination with respect to serum/blood, is surely intriguing.The first evidence of an increased production of NO and derivates in IPF lungs was published in 1997, when Saleh and coauthors reported a significant overexpression of iNOS in inflammatory cells and alveolar epithelium associated with relevant production of nitrotyrosine compared to healthy subjects . A higheConcerning the association with pulmonary functional parameters, an inverse correlation between CaNO values and FVC and DLCO has been repeatedly observed, suggesting a potential value of this biomarker in the monitoring of disease progression ,42,44,45To date, no studies have been conducted to investigate the impact of antifibrotic treatment on CaNO values and if extended eNO analysis may be useful to predict or to quantify, the response to therapy in IPF patients.The only data available for extended FeNO analysis in granulomatous ILDs concerns two distinct disease entities: sarcoidosis and hypersensitivity pneumonitis (HP). Many studies have investigated the potential role of NO in the pathogenesis of sarcoidosis, reporting conflicting result. Although an overexpression of iNOS was demonstrated in sarcoid granulomas, significantly different than in other granulomatous disorders ,73, measMultiple-flows eNO analysis was conducted in three studies ,47,48, bRegarding HP, very few and heterogeneous data are available, as eNO evaluation was conducted with different methods and in different phases of diseases (acute and chronic). Only two studies specifically assessed FeNO in patients with HP. Ojanguren et al. measured conventional FeNO before and after a specific inhalation challenge (SIC) to evaluate its potential role in diagnosis, reporting negative results . Only onExtended eNO analysis has been specifically investigated in asbestos-related lung disease. A significant increase of CaNO values, but not of FeNO and bronchial NO levels, in patients with asbestosis was firstly reported in 2007, with the authors suggesting a correlation between asbestos-related alveolitis and CaNO . SimilarEosinophilic pneumonia is a rare inflammatory lung disorder characterized by a massive infiltration of eosinophils in alveolar spaces and interstitium; clinical aspects and radiological features are not very specific and usually BAL cellular count analysis is necessary to confirm diagnosis . ConsideThe potential value of eNO in the differential diagnosis of IPF is surely intriguing, but still largely unexplored. Few studies have been specifically designed to investigate the potential value of eNO parameters in the differential diagnosis of ILDs ,8,51,55.In summary, extended eNO assessment seems to have the potential to be implemented in the diagnostic pathway of ILDs, particularly to discriminate CTD-ILDs and EP, mainly due to its reproducibility, noninvasiveness and relatively low cost. However, multicenter and prospective studies are needed to confirm these findings and better evaluate its potential utility in this fiePulmonary arterial hypertension (PAH) is one of the most common complication of ILDs and is invariably associated with a significantly worse prognosis, quality of life and mortality ,86,87. AMany studies have investigated eNO behavior in patients with obstructive sleep apnea (OSA), reporting slightly, but still significantly higher levels of FeNO and J’awNO and, conversely, reduced CaNO values than healthy controls ,95,96. IThe association of lower-lobe fibrosis with upper-lobe emphysema was first proposed as a distinct clinical entity and defined as combined pulmonary fibrosis and emphysema (CPFE) in 2005 . To dateA higher prevalence of lung cancer in patients affected with ILD with respect to the general population has been repeatedly reported. However, it is still a matter of debate if ILD (particularly IPF) increases per se the probability of lung cancer development or is just related to the overlapping of risk factors associated with both diseases  ,106,107.In this review, we report the published evidence concerning the potential value of extended FeNO assessment in the management of patients with ILDs. Multiple-flows FeNO analysis is surely an interesting technique that could allow respiratory physicians to obtain a reproducible and inexpensive biomarker for the management of diffuse lung diseases. The standardization of technique, and the endorsement of ATS/ERS for extended FeNO as a source of biomarker, have boosted and ameliorated the quality of studies on this topic, although they are still few and monocentric. Among ILDs, many specific disease entities may show a progressive clinical course similar to IPF, leading to chronic respiratory failure and death in a few years. However, no biomarker has been widely accepted in the clinical practice in both diagnostic and prognostic estimation. In this field, CaNO has demonstrated an intriguing potential in discriminating idiopathic ILDs from CTD-ILDs and in estimating survival and disease progression in terms of FVC deterioration, suggesting its possible implementation in the clinical management of IPF due to its reproducibility, repeatability and noninvasive nature. However, these promising findings need to be furtherly confirmed by larger, prospective and multicenter studies to better investigate the dynamics of eNO parameters throughout the clinical course of disease and to evaluate the influence on CaNO played by comorbidities and antifibrotic treatment.


Inorganic polyphosphate (polyP) is a ubiquitous polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. In prokaryotes and lower eukaryotes, both the presence of polyP and of the biosynthetic pathway that leads to its synthesis are well-documented. However, in mammals, polyP is more elusive. Firstly, the mammalian enzyme responsible for the synthesis of this linear biopolymer is unknown. Secondly, the low sensitivity and specificity of available polyP detection methods make it difficult to confidently ascertain polyP presence in mammalian cells, since in higher eukaryotes, polyP exists in lower amounts than in yeast or bacteria. Despite this, polyP has been given a remarkably large number of functions in mammals. In this review, we discuss some of the proposed functions of polyP in mammals, the limitations of the current detection methods and the urgent need to understand how this polymer is synthesized. Arthur Kornberg and Igor Kulaev spent a large part of their careers working on inorganic polyphosphate (polyP). Convinced of the fundamental importance of this ‘forgotten polymer', they and their teams developed tools to study polyP in a wide range of organisms from bacteria to mammals ,2. TodayDictyostelium discoideum, two PPK activities have been originally reported, one with sequence similarity to the bacterial PPK1 that was acquired through horizontal gene transfer  radiolabelling, are therefore required. In mammals, polyP was first reported in 1963 and its characterization was performed using a set of biochemical assays. Nowadays, besides these classical assays, the semi-quantitative detection of polyP is achieved using microscopy after staining with fluorescently tagged proteins or fluorescent dyes. These approaches are now reviewed and discussed.The high abundance of polyP in micro-organisms facilitates its detection and quantification. Mammalian cells possess much lower amounts of the polymer and more sensitive methods, such as [32P] allows detection of lower polyP amounts, it also requires the use of defined media with low phosphate, a condition that, as demonstrated in micro-organisms, favours degradation of polyP rather than its synthesis. To overcome these drawbacks, other approaches were later developed and optimized: extraction procedures involving acidic phenol or guanidine isothiocyanate, and specific enzymatic assays reducing the need for the polyP extract to be of the highest purity. The bacterial exopolyphosphatase Ppx1 is now routinely used to degrade polyP in vitro. The orthophosphate residues released are then quantified by standard assays such as the malachite green. Alternatively, the reverse reaction of the polyphosphate kinase Ppk1, consisting of the transfer of phosphate from polyP to ADP, generates ATP that can be quantified with the sensitive luciferin/luciferase assay. Finally, the use of polyacrylamide gel electrophoresis (PAGE) is critical in the definitive identification of polyP, as it allows the detecting of a ladder like appearance, consequent of the stepwise increases of polyP's polymeric nature. PAGE is also essential for the estimation of the chain length of the polymer. The omission of urea from the PAGE recipe  labelling. The most widespread dye used to visualize polyP is DAPI, which is better known for its nucleic acid binding properties. However, the ability of DAPI to bind to a large variety of molecules may hamper the detection of low amounts of polyP as in mammalian cells. Recently, more specific fluorescent dyes found to bind polyP (JC-D7 and JC-D8) allowed its visualization in living mammalian cells -ATP to improve sensitivity. Therefore, the quest to find the mammalian polyP synthase is still open and for the health of the research field must be accomplished soon. Only then might we be able to confidently say not only where Wally is, but also what are its actual physiological functions.The discovery of the polyP synthase in mammalian cells will be of instrumental importance to validate all these observations. This will require classical biochemical fractionations and assays likely requiring the use of radiolabelled [


Entropy plays a key role in the self-assembly of colloidal particles. Specifically, in the case of hard particles, which do not interact or overlap with each other during the process of self-assembly, the free energy is minimized due to an increase in the entropy of the system. Understanding the contribution of entropy and engineering it is increasingly becoming central to modern colloidal self-assembly research, because the entropy serves as a guide to design a wide variety of self-assembled structures for many technological and biomedical applications. In this work, we highlight the importance of entropy in different theoretical and experimental self-assembly studies. We discuss the role of shape entropy and depletion interactions in colloidal self-assembly. We also highlight the effect of entropy in the formation of open and closed crystalline structures, as well as describe recent advances in engineering entropy to achieve targeted self-assembled structures. S), a macroscopic property of systems that was discovered by Clausius [W is the number of (quantum) states or volume in phase space accessible to the system.The second law of thermodynamics leads us to the state function entropy , and vibrational. Translational entropy is related to arrangements possible due to translational degrees of freedom, and rotational entropy is associated with the unique ways in which the particles can rotate/orient, while vibrational entropy reflects the vibrational motions of particles . This caSelf-assembly, also called ordering, is a well-known phenomenon where individual particles in a system spontaneously organize to form ordered structures . This prtrations .Like any other spontaneous process, self-assembly is driven by the minimization of the free energy of the system . The chaEquation , where ΔTherefore, a system can lower its free energy either by decreasing the internal energy or increasing the entropy. For ordering phase transitions , one can then rationalize that a spontaneous phase transition takes place if the internal energy of the system is sufficiently lowered to outweigh the loss in entropy. While this intuitive interpretation suggests ordering transitions as energy-driven, the opposite has been argued in that the ordering phase transitions are in fact entropy-driven . TherefoIt is possible to observe the formation of self-assembled structures in biophysical processes, including in DNA assembly , proteinColloidal particles are small solid particles whose dynamics in suspension are driven by thermal energy fluctuations . They arS simply to the logarithm of S to the logarithm of For colloids, the application of the definition of entropy in terms of the logarithm of a volume in phase space  . Tuning The shape of colloidal particles influences packing, directionality, selectivity, connectivity, and dynamics in self-assembly processes . ExperimFurthermore, branched colloidal nanoparticles, such as octapod-shaped nanocrystals, can self-assemble to form three-dimensional superlattice structures under proper conditions . Loudet Monte Carlo (MC) and Molecular Dynamics (MD) simulations can be used to predict self-assembled structures that maximize the entropy of a system under given conditions . For insSimulations have also shown that hard convex polyhedra self-assemble into different crystalline structures due to the directional entropic forces acting between the particles ,68. The Another example of a purely entropically-driven self-assembly process is the ordered arrangement that arises in colloidal systems of large spherical particles when smaller particles (depletants) are added, a process known as the depletion effect . Depletiu) is described by Equation u=−ρQ) when the colloidal system is diluted [Mathematically, the relationship between the change in available volume and the free energy of the system can be defined by Equations (5) and 6), where Equation F=−kN is the number of particles in the system, Q, which leads to a lowering of the free energy of the system, per Equation  and nonadsorbing polymer molecules. Xie et al. [Using depletion interactions, Barry et al.  demonstre et al.  studied e et al. , Kemp ete et al.  showed tEntropy also plays an important role in the formation of open colloidal lattices , which cMC simulations of systems composed of DNA-coated colloids at lower pressures showed that these particles self-assemble, forming floppy and open crystals instead of compact ones, due to the higher vibrational entropy that these arrangements provide . Moreovei in the ensembles for the model and target structures, respectively. A further examination of Equation (i for both model and target structures are the same, Relative entropy (a system . Srel caEquation , where pEquation  shows thThe optimization of CG models is carried out by minimizing m system . The conm system , implicim system , and pepm system .Inverse design is a class of computational modeling techniques utilized to discover suitable pair interactions ,103,104 When utilized to determine the optimal pair interactions, inverse design techniques rely on the minimization of the relative entropy (ructures . Inverseructures .The implementation of this technique for determining pair interactions led to the formation of several two-dimensional target lattices, including rectangular Kagome, triangular honeycomb, and octadecagonal star binary , and thrWhile this technique has been successfully used to obtain pair potentials that favor target structures, some of its limitations include the requirement of specifying relevant competing structures and the fact that a target structure that is formed in the ground state might not be formed when the initial fluid state of the particles is cooled from higher temperatures . To overUnlike the usual approach of tuning the pairwise interaction potentials between the particles, tuning the particle shape is a recently developed inverse design method of engineering entropy to produce equilibrium structures via self-assembly . In thisIn this work, we discussed the importance of understanding the role of entropy in ordering transitions in colloidal systems. We also described the ways in which control over the shape and size of colloidal particles self-assembles them to form useful structures with suitable properties for applications in technological and biomedical domains. We described the concept of relative entropy and its usefulness for inverse design methods in achieving optimal particle shapes and desired self-assembled structures. However, experimentally synthesizing particles of different shapes at a high precision is challenging. Moreover, tuning entropy is quite infeasible in experiments, but tuning enthalpy or inter-particle interactions is feasible. However, the study of entropically-driven self-assembly processes is a field that has recently gained attention, as computational resources and knowledge evolve to allow simulations of colloidal systems of particles with unique shapes, and under new conditions. The entropy driven self-assembly has been able to generate complex structures because of the meticulous selection of particle shapes, sizes, and concentrations. In any self-assembly process, the use of depletion interactions and shape entropy together may have a greater ability to form structures having more complexity and variety. In those self-assembly processes, where the energy and entropy both play important roles, it is the entropy that often determines the most probable structures among many competing structures. In recent years, theoretical and simulation studies have taken the lead in exploring the effect of entropy in self-assembly processes, and the results obtained from these simulations are being applied to optimize experimental procedures that yield new types of materials with targeted properties, pushing the frontiers of nanotechnology.


Manipulation of the tissue microenvironment has become a promising method to enhance the regenerative abilities of stem cells/progenitors for musculoskeletal repair. The composition of the microenvironment is determined by the resident cells, extracellular matrix, cytokines, and chemokines as well as the biomechanical property and nutrient status. The microenvironment is altered by the homeostasis and degenerative stage of the native tissue. All the alterations of the surrounding host microenvironment will definitely change the biology of the implanted stem cells/progenitors. Thus, a thorough understanding of the stem cell-microenvironment communication would therefore accelerate the success of musculoskeletal repair. In this special issue with the theme “Interaction between stem cells and the microenvironment for musculoskeletal repair,” we are very pleased to present the 11 accepted papers (7 research articles and 4 review articles) in which how the microenvironment affects the stem cells/progenitors was well investigated.in vitro, Dr. Ni's group used 20 IU/ml erythropoietin combined with the cyclic mechanical stretch ; the underlying mechanism may be associated with the activation of ERK1/2 signaling pathway. Dr. Huang's group found that 10 mM strontium (a trace element in the bone tissue) was able to promote the osteogenic gene expression and mineralization of human BM-MSCs and placenta-derived MSCs. Interestingly, using the exosomes derived from the osteogenically differentiated adipose-derived stem cells (ADSCs), obvious enhancements in the osteogenic differentiation of ADSCs were recorded. Dr. Yang and colleagues further determined that this enhancement was related to the upregulated (201) and downregulated (33) exosomal miRNAs derived from the osteogenically differentiated ADSCs. Hence, beyond the resident cells, the bioactive compounds, and the biomechanical stimuli, exosomal miRNAs probably became a new target to enhance the osteogenesis of endogenous and exogenous stem cells/progenitors.Bone marrow mesenchymal stem cells (BM-MSCs) play a central role during the process of bone modeling and remodeling; yet, their biology is regulated by the cytokines, biomechanical stimuli, and other types of cells. Increased B lymphopoiesis was found in mouse with osteoporosis , which cvs. senescent NP cells, which may assist the development of novel biological methods to treat degenerative disc diseases.During the cause of intervertebral disc (IVD) degeneration, gradual changes in the disc morphology, matrix composition, and microenvironment have been observed , 3. In tin vivo due to the heterogeneity and lack of specific markers and suggested that the mechanobiology of TSPCs and its role in tendon development, growth, repair, and pathology need to be better clarified.The tendon is to transmit muscular forces to the bone, permitting joint motion and subsequent body movement . It was in vivo, in clinics, the expected outcomes of the intrathecal administration of neurotrophic factors alone was hard to achieve. Pawlukowska et al. performed a clinical study using the autologous lineage negative (Lin−) stem cells to treat ALS. The authors thought Lin− stem cell-based therapy would be a reasonable and promising alternative for classic ALS treatments because the Lin− stem cells could produce the trophic support for the host's neurons, stimulate the secretion of neurotrophins (NTs), and differentiate into oligodendrocyte progenitor cells or neurons. In this article, the authors completed a clinical trial to assess the impact of intrathecal administration of bone marrow Lin− stem cells in 32 patients suffering from ALS on articulation; it was demonstrated that 6 patients achieved the improvement of articulation after 28 days, 23 patients remained stable, and 3 deteriorated. Although some valuable findings were observed, several limitations should be acknowledged such as the small number of patients, the lack of control group, and a short observation period.The interaction between stem cells and local microenvironment goes in both directions. Not only the microenvironment can impose on the fate of stem cells, but also stem cells can positively affect the local microenvironment of injured tissues. In Fang's study using a rat peripheral nerve injury model, except from generating neurons, transplanted embryonic spinal cord cells were found to have a regulatory effect on local Schwann cells in the distal nerve and induced them to produce proximal axons to facilitate nerve regeneration. Amyotrophic lateral sclerosis (ALS) is a progressive disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control. Due to the low bioavailability and short half-life During muscle regeneration, as reviewed by Dort et al., the spatial recruitment of proinflammatory and anti-inflammatory macrophages is different in addition to their temporal recruitment; proinflammatory macrophages are located close to proliferating satellite cells, while anti-inflammatory macrophages are found close to the regenerating area containing differentiated myoblasts. Depletion of proinflammatory macrophages resulted in impaired muscle regeneration in animal models. The suppression of the switch of macrophage from proinflammatory phenotype to anti-inflammatory phenotype reduced muscle fiber growth but did not affect the clearance of necrotic tissues. At the cellular level, proinflammatory macrophages promote myoblast proliferation and inhibit differentiation, while anti-inflammatory macrophages inhibit myoblast proliferation and stimulate their differentiation and myofiber growth. Direct coculture of macrophages also promoted proliferation and inhibited apoptosis of myogenic cells. Altogether, these findings suggest that different subsets of macrophages have complementary roles in the regulation of satellite cell/myoblast function, myogenesis progression, and optimal muscle regeneration.In summary, the published articles in this special issue add new perspectives to the understanding regarding the interaction between stem cells/progenitors and the microenvironment for the bone, intervertebral disc, tendon, muscle, and nerve regeneration. We hope that all these basic and clinical studies will be helpful for the development of stem cell-based therapies for musculoskeletal repair. Finally, we would like to express our gratitude to all the authors, the reviewers, and the editorial board members of this journal for their contribution and assistance to make this special issue successful.


Bio-neuronal led psychiatric abnormalities transpired by the loss of neuronal structure and function (neurodegeneration), pro-inflammatory cytokines, microglial dysfunction, altered neurotransmission, toxicants, serotonin deficiency, kynurenine pathway, and excessively produced neurotoxic substances. These uncontrolled happenings in the etiology of psychiatric disorders initiate further changes in neurotransmitter metabolism, pathologic microglial, cell activation, and impaired neuroplasticity. Inflammatory cytokines, the outcome of dysfunctional mitochondria, dysregulation of the immune system, and under stress functions of the brain are leading biochemical factors for depression and anxiety. Nanoscale drug delivery platforms, inexpensive diagnostics using nanomaterials, nano-scale imaging technologies, and ligand-conjugated nanocrystals used for elucidating the molecular mechanisms and foremost cellular communications liable for such disorders are highly capable features to study for efficient diagnosis and therapy of the mental illness. These theranostic tools made up of multifunctional nanomaterials have the potential for effective and accurate diagnosis, imaging of psychiatric disorders, and are at the forefront of leading technologies in nanotheranostics openings field as they can collectively and efficiently target the stimulated territories of the cerebellum (cells and tissues) through molecular-scale interactions with higher bioavailability, and bio-accessibility. Specifically, the nanoplatforms based neurological changes are playing a significant role in the diagnosis of psychiatric disorders and portraying the routes of functional restoration of mental disorders by newer imaging tools at nano-level in all directions. Because of these nanotherapeutic platforms, the molecules of nanomedicine can penetrate the Blood-Brain Barrier with an increased half-life of drug molecules. The discoveries in nanotheranostics and nanotherapeutics inbuilt unique multi-functionalities are providing the best multiplicities of novel nanotherapeutic potentialities with no toxicity concerns at the level of nano range. Psychiatric disorders (neurodegenerative diseases) occur after a series of intriguing incidences such as inflammatory dysfunctions and increased neuroinflammation  The attitudes and behaviors concerning one's illness are products of deduced practices to commemorate, and therefore, it can be affected by cognitive dysfunctions. To understand the mental states of other, it is necessary to replay upon "to see ourselves as others see us". This improves the abilities to know the mental statuses  of others. Therefore, the correct outlook to moody alteration in oneself depends on the abilities to reveal upon personality as a prospective, and that is, the priority of the theory of mind. Insight has to think of as a relational concept that serves as the light of understanding into something i.e. understanding illness, pathological nature traits, specific symptoms, current syndrome, and social difficulties, etc. Finally, there are many pieces of evidence for elucidating the role of neuroinflammation in various psychiatric illnesses as abnormal neurotransmission, serotonin deficiency, because of the changes in neurotransmitter metabolism, pathologic microglial cell activation, impaired neuroplasticity, and structural and functional brain changes responsible for cognition and emotional behavior -), an influential oxidant, capable to damage the biological molecules are among the main damaging constituents that exist in brain functioning. As in the necrosis process, tumor necrosis factor-alpha (TNF) -α medicate cross-linkages between neurons and astrocytes for further engagements Toxicants enter cellular, molecular, and inflammatory pathways to start the initiation of processes for the origin of diseases. These foreign objects damage the brain's cellular process. These destructions in the functioning of neurotransmission processes and protein networking cycles propagate the pathogenesis of neuropsychiatric diseases. To encounter these undesirable difficulties in the cell's physiology, the cytokines, an interface regulator for interactions and communications between cells, stimulate the neuronal defense fortresses of immune cells to secrete anti-inflammatory cytokines for healing the prolonged pain a) Impact of oxidative stress on the neuron: Lake of sufficient oxygen (O2) in the cell causes reduced production of ATP. Such dysfunctions force neurons to reduce their activities, and as a result, the neurodegenerative processes get started. But the interrelated mechanism is not still clear and how it happened, also not yet discovered. The free radicals and non-radical derivatives are there for signal sensitization in the brain to deal with oxidative stress whenever redox signaling goes awry . The ribonucleic acid (RNA) oxidation is an underappreciated cause of oxidative stress Figure  To disclose the further distinct biochemical proceedings, the nanotheranostics must urgently employ for the observations of oxidative stress in the brain and disease 2O2) by converting it to (O2) and water (H2O). Oxidative stress, the deleterious condition, occurs whenever ROS intensities go beyond the antioxidant volume of a cell. As the neurons take part chemically via neurotransmitters for chemical transmission, and neuromodulation  Tight homeostatic control over the production of ROS in normal cells, containing neurons, is essential for smooth cell regulation. There is a need to detoxify ROS, and biological antioxidants , carotenoids, and ascorbic acid), because these react with most of the oxidants available at the spot. Besides, the antioxidant enzymes  detoxify hydrogen peroxides (Hb)Inflammasomes in neuroinflammation and neurodegenerative diseases: Aggregated host proteins , deposits abnormally within the cell, and near the territories of the cells that initiate neuroinflammation and neurodegeneration Figure The cytotoxic consequences and existence of aggregated misfolded proteins are liable for chronic neuroinflammation and psychiatric disorders c)Cellular and molecular neuroinflammatory pathways and kynurenine pathway induce neurodegeneration: The mechanism of neuroinflammation governed by neurovascular units  perform as a platform to coordinate with pro- and anti-inflammatory mechanisms The mechanism of neurovascular unit dysfunction affected by neuroinflammation and mishappenings as infections. So, the process of neuroinflammation is likely to be a normalization effort in response to any offense affecting the host-defense mechanism gondii. It bases the pathways of these mechanisms on different activation signals and mediators. These activation signals activate cellular phenotypes , and these signaling mechanisms further initiate controlling of the cytokines secretion, misfolded protein mechanism, and infection Figure  The mechanisms of the activation of the phenotypes for responding towards the signals received from the multifaceted neuron-microglial of the CNS, and how they secrete different responding factors to alter the signal of forces, are the key mechanisms to highlight Viruses  normally existed in the brains of diseased persons as having the protozoa Toxoplasma d) Mitochondrial dysfunction, reactive astrocytes, and neuronal cell death: Concerning dynamics of mitochondria governs a lot of important mechanisms i.e. ROS production and sequestrations, ATP production, intermediate metabolism, neurotransmitter generation and degradations, apoptosis, and Ca2+ buffering 2+, K+), tissue repair via angiogenesis and neurogenesis, energy storage, mitochondria biogenesis, defense against oxidative/nitrosative stress, and synaptic modulation 2+-activated K+ channels Figure  The chief causes associated with neurodegeneration are dysregulation of Ca2+ homeostasis and mitochondrial dysfunction 2+ waves and global Ca2+ signaling and soon, thereafter, the mechanism of dysfunction of mitochondria dysregulated Ca2+ related mechanisms responsible for the necrotic neuronal death occurred just because of dysfunctional and dysregulation 2+ concentration in the cells, the discharge mechanism of glutamate, increased concentration of the arachidonic acid, gene alterations and activation leading to cytokine synthesis, synthesis of enzymes involved in free radical production, and accumulation of leukocytes) Identifying these critical functional and structural changes and their mechanisms of action responsible for cell death are still unknown. Shortly, only the nanotheranostics will be the best option for exploring newer diagnostic and imaging techniques to fulfill the existing needs Figure 5. Thus, there is an urgent need to frame such strategies that must have a critical size and ability to remain stable during the cross over mechanisms. The innovation of nanotheranostics resolved this issue To distinguish between mental illnesses, diseases, disorders, or syndromes are the toughest task. There is no scientific process available to differentiate psychiatric illnesses. In the current scenario, no lab test exists in laboratory manuals. The documentation of neurobiological features, neurotransmitter pathways, structural abnormalities on neuroimaging, and genetic predispositions may be helpful in this concern Implementing the novel concept of nanotechnology in neuroscience by using newly designed nanotheranostics and nanotherapeutics are the recent trends and new avenues for treating neurological and mental disorders. Some of them can act all together as nanotheranostics and nanotherapeutics, and such abilities very well appreciated and implemented in diagnosis and therapy Utilization of peptides in neuropharmaceuticals and nanotheranostics for diagnosis and cure of a wide variety of mental illness, disorders, and neuropsychiatry. Designed strategies of nanotheranostics based on peptides are recent developments for the innovations of tools that provide a lot of opportunities for enhancing further understanding to diagnose neuropsychiatric disorders. The tools of neuroimaging and electrophysiology detect the etiology and pathologies of CNS disorders and such efforts enhance the possibilities of exploring the mechanisms underlying inflammation and neurodegeneration Figure 6. The upcoming challenges are there for the search for promising nanocarriers to heel neurological and psychiatric disorders, and their activities must be in the inner part of the CNS Neurological disorders, yet to diagnose fully, need original treatment strategies that remain in concern to heal mental illnesses. The major worries and successes always remain there in the conscious mind because sometimes drug deliveries fail, (unable to reach the site) and diagnosis (molecular imaging available) succeeds or vice versa because of existing multifaceted complications. Therefore, such an unclear phenomenon and an incomplete set of tools always push earlier diagnosis on the back foot. Therefore, the therapeutic option never comes out on both fronts So, there is another concern that arose just because of alterations , along with existing befell in pharmacokinetics and pharmacodynamics of the known therapeutics, mainly directly linked to the route of administration Current therapeutics have a lack of qualities  preferred as inbuilt properties to be present in perfect therapeutics prescribed for the neuropsychiatric illnesses . The efforts for the innovation of novel nanotherapeutics held up in the absence of perfect strategies. Nanomaterial induced nanocarriers and multifunctional nanoplatforms used in their designing might be successful and to consider as a key component in the innovation of nanoformulation with high treatment efficiencies Nanomedicines, in nano-therapy, comprise nanomaterials, nano biosensors, and nanoscale biomaterials to perform as a vehicle and a tool to deliver and release the drugs at a precise specific target. The therapeutics to treat psychological disorders and mental illness The authors also critique the trends in psychiatric neuroprotection and address future trends and challenges by covering the aspects to theoretical frameworks based on psychiatric neuropathology and its applicability Nanobots will restore damaged tissue and neurons in the brain and are capable to excite definite sections of the brain artificially to neutralize depression and anxiety. Inserted probes or derived from nanomaterials to implant in neurons incite the happiest sites inside the brain to balance depressive signs. The intensifying brain illness could efficiently improve by the pre-programmed nanobots included into brains to restore openness, and employed in the development of artificial nano-replacements, promote stem-cells, renewal of faded cells and tissues in vivo imaging, metabolome analysis, and modeling of the central nervous system? It is clear from the reported literature that new nanoparticles assemblies able to create free-scale networking that will perform as artificial neural systems for the healing, repair, and functioning of synapses, diffusion of a neurotransmitter, and mimic synaptic behavior The tiny tools employed to seek space in the interworking of neural networks in the dense brain. Why is nanotechnology considered treating psychiatric illnesses What is the role of neuronal networks in brain functioning, how is it responsible for CNS disorders, and how is any therapeutics to apply if any dysfunction occurred? The most critical aspect is how the brain performs many of its major complicated tasks. We can only explain this via the investigation of neuronal network control in-vivo for most of the neuropsychiatric disorders The communication between complicated architecture (neurons and brain regions) concerns energy minimization, and information transfer In modern-day nanomedicine, there is an enormous need for indispensable tools in disease monitoring and therapy at the nanoscale, and the “engineered” nanomaterials have tuned to a harmonious set of tools as required. Thus, this modality has progressed to gain significant attention in the neurotherapeutics sciences concerning to their versatility of tunes characteristics It represents stress as a sequence of incidences of physical, chemical, and emotional phenomena involving the brain. Mental tension generated this. Stress could be a revolving factor to generate complex diseases. The sequencing of these incidents produces a lot of biological and chemical entities that promote various factors to imbalance the body's equilibrium. Emotional factors and originated harmful chemicals affect the nervous system. Overall, it is tough to distinguish stress and fear, and the same huddle to pinpoint the diseases. A three-dimensional approach needed here for healing the wounds of memory. On one side, there is a need for an exact differentiation of the symptoms to define the psychological imbalance, and the on the other side, there is a requirement of the evaluation requires for a proper nanotheranostics technique to diagnose the nanodomain, that exist in the stress's territory devil or where are the damaged neurons in vivo in the human brain. Mechanically actuated DNA origami robots are potential thought-controlled nanobots that can alter the response to a brain's cognitive state. This benefits the therapeutic control in disorders such as schizophrenia, depression, and attention deficits, which are among the most challenging conditions to diagnose and treat Glial cells are the key component in the brain for responding to the brain's response The symptoms of psychiatric illnesses have similarities with a terrible reputation of unwarranted behavior or cannot say it is just dangerous behavior. If such symptoms like hallucinations, confusion, suicidal thoughts, delusions, depressed mood, rapidly fluctuating mood, memory changes, incoherent thought patterns, elevated mood, addictive behaviors, are there then there is a need for accurate analysis of the symptoms and allied specific patterns of symptoms. Patients with psychiatric illnesses often have impaired insight, which means they have a diminished ability to understand the nature of the disorders. To find out a pattern of mental illnesses and the nature of a particular illness are two unique kinds of situations. Such complications make it even more complicated Because of the inflammation, undesirable damages start at the cellular defense system, which was duly opposed by initiating an immune response to protect from pathogenic infection, to prevent tissue damage, to hinder autoimmune signals within neurons. The fight starts with an imbalance in neuron cells caused by diseases. With uncontrolled inflammation as the prolonged one, it starts chronic diseases . And there are also a lot of diverse phenotypic consequences as dysregulation of immune homeostasis Side effects of drugs, hundreds of metals, particulate, and volatile organic compounds, environmental pollutants, metal particles, and chemicals, can cause cardiovascular diseases, mental illnesses, brain hemorrhage, neurodegeneration, and dysfunction of CNS mechanisms. Ultra-fine particles triggered oxidative stress and neuroinflammation, which further affect the white matter of the neurons in the brain


The developing process ispresented in detail and each simulator was tested and re-tested, beingsubmitted to improvements until their final version. The simulatorspresented are: delivery simulator shorts, Neoprene uterus for postpartumhemorrhage management, and perineal repair simulator. A pilot study wascarried out to evaluate the perception of apprentices through a structuredquestionnaire, using the Kirkpatrick evaluation model. Data weredescriptively analyzed.the respondents (31 apprentices) positively evaluated the simulators,perceiving significant gains in theoretical knowledge, ability to solveclinical problems and decreased anxiety to deal with situations similar tothose simulated.low-cost, handmade simulators are feasible and effective, resulting inpositive learner evaluations. Their availability as open technology allowsthe dissemination of their use. In this context, research and experimentation aimed at teaching goodobstetric practices are particularly relevant for Nursing, considering thehistorical and growing insertion of direct-entry midwives and nurse midwives (MNM)in maternal and neonatal care in Brazil and worldwide.Implementation of good obstetric practices and prevention of maternal and newbornmorbidity and mortality are the focus of obstetric care. Thus training and educationof health care providers towards better intrapartum practices and effectiveemergency management are key-points to improve care. In thiorldwide..Since the 1970s, the Pan American Health Organization and the World HealthOrganization (PAHO/WHO) have described the roles and activities of MNM, which coverdirect care, management, education and research. A report examining the currentstate of Midwifery and Obstetrics in 73 low- and middle-income countries, includingBrazil, presented in 2014 by the United Nations Population Fund (UNFPA) incollaboration with the WHO and the International Confederation of Midwives (ICM),reports that urgent investment is needed to improve the quality of obstetric care toprevent about two-thirds of all maternal and newborn deaths - which would savemillions of lives each year. The countries selected for the report  are responsible for 96% of all global maternal deaths, 91% offetal deaths and 93% of newborn deaths. The goals include adequate access for womento midwifery services; high-quality primary care and the possibility of transfer tonext level when needed; increased availability of MNM and its beneficialinterventions for the mother and baby dyad; and strengthening MNM localassociations.Given the growing recognition of MNM role in the implementation of good obstetricpractices, aiming to improve quality of care and prevent health problems withrelevance from the public health perspective, new challenges associated withtraining and continuing education of these Nursing providers are highlighted.Therefore, studies addressing simulation-based Nursing education demonstrate itspotential in enabling the student to deal with situations of anxiety and stresstypical of nursing clinical practice. Besides aspects of theoretical knowledge,technical skills and critical thinking, students may experience emotional, spiritualand ethical issues regarding the care for patients and their families, involved inthe context of nursing simulation.. When teaching undergraduate students, not all interventions can beperformed autonomously by them (such as emergency situations), so the simulationeducational activities provide the opportunity to experience events that would notbe possible in real-world settings(-). Simulation environments replicate a controlled clinical scenarioallowing detailed observation of students in action, feedback and repetition as manytimes as needed without any harm to patients.In Nursing teaching, simulation-based educational practices have been reinforced inthe national and international literature, as it allows an ethically appropriateapproach and promotes patient safety, considering that the first experience of carewill not be carried out with a real patient. When tsettings-6. Sipatients.. Although the quality of studies is heterogeneous and with severaldifferent indicators, evidence has shown that simulation-based teaching is effectiveand leads to better and more lasting results than traditional teaching. Its use can improve clinical, technical, communication, and teamworkskills, improve performance and reduce errors. There is evidence that simulation-based medical education can improveboth learning and patient care, as well as the clinical practice and still have apositive effect on public health.The use of simulators and simulation environments for teaching health professionalsis well established in the literature,7-8aching,. Its use errors. There health,., mostly obstetric emergencies such as shoulder dystocia(-), postpartum hemorrhage(-), pre-eclampsia and eclampsia(-). There is also evidence on simulation to enhance surgical skills suchas suturing of vaginal and severe lacerations. A 2014 literature review showed that after simulations, it waspossible to observe an increase in knowledge as well as technical, communication andteamwork skills. A 10-year follow-up study of a simulation-based training for shoulderdystocia management identified an increasing in the number of diagnosis and adecreasing in the number of neonatal brachial plexus lesions. At least one study conducted in Mexico was able to demonstrate that the implementation of a continuingeducation program including simulations has successfully modified obstetric practicewith better results in terms of good practices adoption. Another training programwith simulation in Tanzania showed a 47% reduction in postpartum blood transfusionrates.Particularly in Obstetrics teaching, the use of simulators and simulationenvironments has been studied in various settings, mostlydystocia-12, pmorrhage-18, pclampsia-17. Terations. A 2014k skills. A 10-y lesions. At leaonrates.. With well-established learning objectives, the use of low-cost,handmade simulators can be a viable and effective alternative in the teaching andlearning process. Thus, there are several examples of simulators handmade produced andat a reduced cost, both for teaching obstetrics and other specialties/situations. There is no evidence demonstrating that the hyper-reality of thesimulator improves participant learning, the low cost and sometimes even the lowfidelity of a simulator does not seem to represent an obstacle to itsuse(-).Simulation environments and high-fidelity simulators, although proven to be useful,find barriers to their effective use in teaching, the main one being theircost,17. Wcess,-. Thus, etrics-,22 antuations. There  itsuse-21.However, cultural differences in the curriculum of Nursing schools do not allow us tostate with the existing data if the use of handmade simulators is effective forteaching and learning, requiring further study. Additionally, studies in theBrazilian setting addressing low-cost simulators in the context of Obstetricsteaching were not found.Considering this broader context, the objectives of this article are: to describe thecreation and use of handmade simulators for teaching Obstetrics and to present theresults of a pilot study on the use of handmade simulators as educationaltechnologies, through the perceptions of nursing professionals (MNM), obstetricsresident physicians and undergraduate students (apprentices) who participated inclasses using the simulators.This is a cross-sectional pilot study to assess apprentices’ perception about the useof low-cost handmade simulators for Obstetrics teaching, based on the hands-onteaching experience of the developers. The scope of the analysis is characterized asa study to improve the quality of health care, through the improvement of teachingand learning of health professionals. Thus, the SQUIRE guidelines for publicationsof this type were adopted. The methods section will be discussed in two stages: theprocess of developing the simulators and the methods used for the pilot study.http://saudesimuladores.paginas.ufsc.br/.The simulators were created or adapted (based on ideas suggested through panels ofexperts and/or available on the Internet) and made by the authors, based oneducational needs identified in hands-on training workshops for professionals andstudents of Midwifery and Obstetrics offered systematically since 2014. Allsimulators have a Creative Commons license and are available to be reproduced oradapted in other services on the website The simulators presented in this article are: (i) Birth simulator shorts with a doll;(ii) Neoprene uterus simulator for the management of postpartum hemorrhage; (iii)Simulator of perineal tear repair.. It should be noted that the face and content validity depends on thelearning objective. A piece of curvy fabric (synthetic leather), for example, doesnot have the appearance of human skin, but with the proper assembly may be valid forsuturing training. The structure of the simulation is considered to be a process,not a product, and the simulation training needs strategic planning in order to beeffective.The process of setting the simulators began with the identification of an educationalneed observed in a real-world training context. From this need, there was a seek foralready existing alternatives, through the search of previous simulators. The searchsources were films and websites with content on Obstetrics and Obstetrics training,in addition to the constant exchange of ideas and suggestions from peers. Based onthis search, the feasibility of a simulator was evaluated and, when it seemedfeasible - in terms of cost, the possibility of material acquisition andmaintenance, size -, a prototype was developed and tested. The tests were madethrough expert evaluations . The aimwas to establish face  and content  validity,24. Iffective.On many occasions, tests have determined changes in the prototype. This was redoneand retested as many times as was necessary. At the time we obtained a version ofeach simulator that was considered appropriate, they were considered finished andavailable for implementation. Even after implementation, it must be considered thatthe simulators should continue to be tested and may undergo improvements andmodifications. We hope that the simulators are recreated and adapted to differentrealities, with citation of the original authorship. and emergency breech vaginal birth. The rigid plastic or rubber birth simulators  do not allow these maneuvers to be performed adequately and make it difficultto train the provider/birthing woman interaction, including the dynamic effects ofthe birthing woman movements on dystocia management. The model consists of aLycra shorts with a hole and zigzag stitching that willsimulate the maternal perineum at birth. The doll used is a toy baby bought inchildren’s toy stores  with a placenta made of crochet, as shown in For the birth simulator shorts with a doll, the educational need identified was theneed for training maneuvers that require changing the woman’s position, such as forshoulder dystocia and emeal birth. The riOne student or actress wears the shorts and may experience the birth, while anotherstudent may attend the birth, identify and manage dystocias. This simulator allowsthe training of skills and attitudes related to normal delivery care, resolution ofdystocia and obstetric complications . It was also used for the discussion of models of careand communication skills, as it allows apprentices to play the birthing person role.It is a low-cost solution with a final cost of approximately USD 26.91  and allows the presentation andsimulation in various environments - schools, pregnant women groups, andprofessional training environments.. There are simulators on the market, even tested with excellentresults. However, although they are considered low-cost (compared to others inthe market), they are not affordable in our reality.Regarding the Neoprene uterus simulator, the identified educational need was skillsand attitudes training for the management of postpartum hemorrhage, which is a highprevalence condition and one of the leading causes of maternal deaths inBrazil. There results,28. H. Also, when the instructor’s hand compresses the ball internally, thereturn of uterine tonus after treatment is also simulated. After the initial tests,the same model presented in .A uterus simulator made of Neoprene fabric (synthetic polychloroprene elastomer) wascreated in a domestic sewing machine. Initially, a Neoprene model was made with asemi-inflated plastic ball insert that allows simulating the uterine contractilitywhen squeezed. This model can be used to simulate the diagnosis of uterinehypotonia, performing abdominal uterine massage, bimanual uterine compression. Also, nted in .per unit. Thissimulator was validated by 18 specialists in the field (doctors and midwives) whoconsidered that it replicates the anatomical structures and tactile sensation of theabdominal and bi-manual uterine compression.The production cost is approximately USD 17.63 pression.The simulator can be used on a workbench to visualize/feel/train the technique ofmassage and balloon insertion. It also fits to be placed in shorts similar to thebirth simulator shorts - adapted with Velcro - for the simulation of postpartumhemorrhage with an actress as the patient, which allows training of the variousmanagement steps, communication with the patient and teamwork. This simulationperformed in a real environment (labor and delivery room) allows for recognizingdifficulties  and search forsolutions. and many professionals are not familiar with this technique.Perineal tears can occur during vaginal births, usually spontaneous lacerations withvarying degrees of severity, most of which are superficial without need fortreatment. Some second-degree tears  require suturing, which can be performed by both nursingprofessionals and doctors. The best technique for suturing second-degree tears(involving skin and/or mucosa and muscles) is continuous suturing in all planes witha single suture thread and man. The obstetrician is technically responsible for this repair, but manyresidents and trained professionals feel insecure and unable to perform thesuturing, due to its rare need and their little exposure to the procedure duringtraining.Severe tears (third and fourth degree) reach the external anal sphincter and rectalmucosa, respectively. They are less frequent and have a potential for permanentsequelae and pathologies if not properly repaired. The obsuturing, due tord and 4th-degree lacerations, asshown in Thus, regarding the suturing simulator of second-degree and severe perineal tears,the educational need found was therefore training for postpartum perineal repair.Several models were tested and two different simulators were proposed. A foam modelfor training second-degree perineal lacerations and a model with a male condom,bovine tissue, and beef for 3To evaluate the simulators, three different groups of learners completed a structuredquestionnaire: undergraduate medical students, gynecology and obstetrics residentdoctors, and nurse midwives, who attended obstetric emergencies training using thesimulators. The only inclusion criterion for answering the questionnaire was havingparticipated in a class with simulation, using at least one of the simulators. Thesimulators have been developed and employed in hands-on simulation workshops since2014 and the study was conducted in March 2019.Likert scale. Thequestionnaire also presented an open-ended question on the use of acquiredknowledge, skills, and attitudes in real-world situations and an open space forobservations.Participants were invited to participate voluntarily and without any benefits orharms to their activities. All volunteers were informed about the study aims andsigned the Informed Consent Form. A semi-structured questionnaire was created by theauthors specifically to evaluate the simulators. It was anonymously answered by therespondents themselves and contained questions to assess the reaction,self-perception of learning and behavior, in addition to questioning about theimmersion in the clinical situation that the simulator allowed. It consisted ofeight closed-ended questions using a . The popularity of this model is due to the fact that it results in awell-defined system for demonstrating the results of an intervention ortraining. Although it is subject to criticism , it simplifies the complex process of trainingevaluation. To measure the impact of the intervention, questions were formulatedbased on the Kirkpatrick’s assessment model, which proposes 4 levels of assessment:reaction , learning, behavior (whichknowledge and/or skills acquired through the training were applied to the learner’swork and/or resulted in improved performance) and outcomes (-). The closed-ended questions sought to evaluate the reaction(satisfaction and immersion of the learner in the simulation), the learning, behavior  and the outcomes . The open-ended question sought to assess at the qualitative levelwhether and how the learners used the learnings obtained from the simulations inreal situations, to analyze both behavior and outcomes levels of Kirkpatrick’smodel.There are several models for the evaluation of the simulation process effectiveness,and Kirkpatrick’s model is one of the most used. This model, initially proposed bythe author in the 1960s, is widely accepted and used since then,31. Taluation. To meaectives)-31. TThe quantitative data were transcribed to a spreadsheet and analyzed usingdescriptive statistics. Since this is a pilot study, no hypothesis testing wasemployed and sample size calculation is not applicable., using the thematic modality to reveal the meaning of the experiencefor the participants. We developed the stages of pre-analysis, material exploration,treatment of the results and interpretation, and then proceeded: 1) data ordering,which included transcription, rereading and organization of the answers; 2)categorization of the information, after exhaustive and repeated readings of thetexts, to define the category of analysis; and 3) The third stage was developedthrough the observed relationship between the interpretation of answers and theevaluation issued by the participant, which allowed the design of the followingcategory of analysis: “The training helps the care provider to feel safer and moreconfident”.The qualitative analysis of the open-ended question had a complementary role to thestudy and was guided by the content analysis method, using Ethical requirements applicable to research involving human beings were respected,and the project was approved under the record number 71272017.4.0000.0121 of theEthics Committee of the Federal University of Santa Catarina, in compliance withResolution 466/2012 of the National Health Council/Ministry of Health.For the simulators evaluation through the pilot study, 12 gynecology and obstetricsresident doctors, 15 undergraduate Medical students and 11 nurse midwives whoparticipated in an obstetric emergency training using the simulators were invited toparticipate. We obtained 31 replies to the questionnaire: 10 resident doctors, 12undergraduate medical students, and 9 nurse midwives, with an 81.58% response rate,similar in all groups.The mean age of all respondents was 29.68 years (SD 6.36). Among midwives  the mean age was 35.44 (SD 7.45) years.Among residents, the mean age was 28.7 (SD 3.56) years and among medical students26.17 (SD 4.24) years. All the medical students were in their fifth year of theMedical School and participated in the simulation less than 6 months ago. Amongresidents, two were in the first year, three in the second year and four in thethird year of training. First-year residents participated in the simulation lessthan 6 months ago. Those from the second and third years have been participating inthe simulations since the beginning of their residency, in a repeated and systematicway. MNM have, on average, 8.89 (SD 8.65) years of experience and all participatedin the same emergency course with the use of simulators in the 24 months prior tothe questionnaire application.The questionnaire responses are summarized in the training helps theprofessional to feel safer and more confident, reducing anxiety and improvingperformance.The open-ended question about the use of the acquired knowledge in real situationswas answered by midwives and medical residents. According to the answers, whensimulated situations occur in the clinical practice, After the class with the simulator, I had to manage a post-partum hemorrhagecase and could confidently follow the steps (Resident of the 2nd year);I alone solved a hemorrhage case (Resident of the 3rd year);Before the class with the simulator, I had too many difficulties even toidentify the planes. After the class I feel safer and more confident. Theresidents, after training, have already taught some doctors in the service thenew techniques of suturing (Resident of the 2nd year); I wasfaced with a shoulder dystocia case and felt calmer to resolve(Resident of the 3rd year); I acquired greater agility to resolve bleedingcases (nurse midwife).In the open space for observations, at least three respondents indicated that theyhave used or intend to use low-cost handmade simulators for the training of otherprofessionals or in groups of pregnant women. This finding indicates that one of theinnovations that the models bring, besides the security and confidence with therealistic simulations, is the possibility of an easy reproducibility in theircontexts, making the knowledge more democratic., the same happens for low-cost/artisanal simulators.We aimed to describe the developing process of low-cost handmade simulators thatallow apprentices to acquire knowledge, increase skills and train attitudesregarding various obstetric procedures. An evaluation of the perceptions of studentsand professionals who used the simulators in a real-world training context was alsocarried out, adopting an evaluation methodology for simulators that allowed toaddress the reaction of the learners. In our sample, all those who answered thequestionnaire were satisfied with the simulation. Apprentice satisfaction is oftenhigh in simulation environments,34, tmulators,19.. The use of simulators presents several advantages such as thepossibility of repeating the procedure, correcting errors, and perceiving thedifficulties . This is expected to improve performance in real-world situations.The self-perception of the learning acquired was positive, with all respondentsconsidering that the simulator class increased their theoretical knowledge andskills. A fact also observed in several studies, especially when the evaluation isperformed by the apprentice themselves-,34. Tocedure). This i. The differences between low- and high-cost/high fidelity simulatorsare not established and warrants further studies.Currently, the evidence is quite consistent to state that simulation-based trainingin Obstetrics improves knowledge and skills. The improvement of clinical andsurgical practices is emerging with great consistency. Improvements in populationaloutcomes are less consistent, but there are some evidence mostly those related toneonatal outcomes,,,20. Ttudies-,28..The evaluation of the hands-on application of the knowledge and/or skills acquired inthe training - behavior and results in the Kirkpatrick classification - was done bythe last three questions of the closed-ended questionnaire and the open-endedquestion. Among the respondents who are currently working in labor and deliveryrooms and maternity services (residents and nurse midwives), most reported that theuse of simulators helped them solve problems. The most cited were cases ofpostpartum hemorrhage, shoulder dystocia and perineal repair. Almost all respondentsalso believe that the course where the simulator was used reduced theiranxiety/stress to deal with a situation like the one presented. A fact that iscorroborated in other studies,,36..As a pilot study, the study scope was restricted to the opinion and experience of theapprentices. Some apprentices considered that, because the simulators were simple,they did not allow the ideal learning. In fact, to allow the student to get involvedwith the simulation, it must be challenging and require effort to resolve. One ofthe questions was whether the simulator allowed the student to feel in a realclinical setting. Eight respondents agreed with this statement. The data presentedhere may indicates that the more advanced the learner, the greater the fidelity ofthe simulator needed for them to feel immersed in the simulation.. Furthermore, the design and nature of the study did not allow acomplete assessment of the 4th level of Kirkpatrick, which is the impact on results, which would need a more comprehensiveanalysis.One of the limitations of the study is the small number of respondents due to itspilot study nature. Additionally, there may be a courtesy bias in the responsesreceived, although secrecy and confidentiality of the information source wereguaranteed. We cannot rule out the hypothesis that precisely those less satisfiedwith the classes and simulations did not answer the questionnaire. Also because itis a pilot study, an objective assessment of knowledge before and after thesimulation was not made, as would be ideal. Furthekpatrick,20, wopen source). The publication ofnew ideas and new simulators and the reproduction and modification of existing onesis allowed and encouraged.The proposal of the project of handmade simulators is that people can access andreproduce the simulators, expanding the resources that teachers who work in thetraining of new professionals have at hand to increase the effectiveness of theireducational strategies. The aim is to freely publish how to make the simulators andkeep them as an open technology (An unexpected finding of the original project and this study was the involvement ofapprentices with the project and handmaking of the simulators, giving suggestionsand actually participating in their development. This involvement favors thedeepening of knowledge, since, to assemble a simulator, it is necessary to accessand put into use knowledge of anatomy, obstetrics, surgical technique, physiology,etc. Additionally, the participation of the apprentices in the development processhas created opportunities to learn knowledge, skills, and attitudes usuallyconsidered extracurricular, but useful and interesting for a richer and broaderprofessional performance, such as research and search for materials, sewing,bricolage in general, creativity in problem-solving, adaptation to low-resourcesituations, etc. Besides, at least three people who answered the questionnairesmentioned that they have already developed or are planning to develop similarsimulators for training, based on the use of those described here. No similar datawere found in the literature, so this is an innovation of the work presented.Further research can expand the knowledge about the development and use of simulatorsfor practical training in Obstetrics, Nursing and other health areas. Specifically,evaluation studies with a larger number of participants, employing othermethodologies that allow assessing not only the learners’ perception but also theeffect on their practices and the concrete results of the training to reduceobstetric and neonatal complications.This study demonstrated the effectiveness of handmade simulators, built by theteachers themselves and at a reduced cost to enhance Obstetrics teaching. It showsthat it is possible not only that the simulators can be low-cost, but that they canbe created by teachers and students, with good results. The use of this type ofsimulators went beyond the improvement in clinical practice, stimulating students todeepen their knowledge and even to develop new simulation environments. It is apioneer study in Brazil and it is expected that the models tested will be replicatedand used in other locations and situations. The study is also expected to stimulatefurther research in the area.The pilot study revealed that the apprentices perceive that simulators favor theexpansion of theoretical knowledge and skills to solve clinical problems, inaddition to the reduction of anxiety to deal with situations similar to thosesimulated. Simulation-based learning is widely recognized in the literature as aneffective method in the context of health professionals’ training, and theavailability of simple, low-cost simulators contributes to broadening access to thisresource for students, teachers, and professionals. Open source technology allowsand encourages these simulators to be reproduced and improved in otherscenarios.


Plants produce some of the most potent therapeutics and have been used for thousands of years to treat human diseases. Today, many medicinal natural products are still extracted from source plants at scale as their complexity precludes total synthesis from bulk chemicals. However, extraction from plants can be an unreliable and low-yielding source for human therapeutics, making the supply chain for some of these life-saving medicines expensive and unstable. There has therefore been significant interest in refactoring these plant pathways in genetically tractable microbes, which grow more reliably and where the plant pathways can be more easily engineered to improve the titer, rate and yield of medicinal natural products. In addition, refactoring plant biosynthetic pathways in microbes also offers the possibility to explore new-to-nature chemistry more systematically, and thereby help expand the chemical space that can be probed for drugs as well as enable the study of pharmacological properties of such new-to-nature chemistry. This perspective will review the recent progress toward heterologous production of plant medicinal alkaloids in microbial systems. In particular, we focus on the refactoring of halogenated alkaloids in yeast, which has created an unprecedented opportunity for biosynthesis of previously inaccessible new-to-nature variants of the natural alkaloid scaffolds. Catharanthus roseus) and listed by the WHO as essential medicines  presents an attractive chassis for refactoring complex biosynthetic pathways of bioactive natural products, thanks to its eukaryotic cell architecture capable of supporting biosynthetic pathways that require significant endomembrane systems  or compartmentalization  grow faster (hours for yeast as compared to months/years for plants), (ii) can be engineered to produce less complex mixtures of plant natural product, and (iii) can be cultivated using more standardized and easily scalable fermentation and downstream processing methods . Buddinglization . Indeed,lization  has insplization . From tyoscapine . Likewisoscapine . In addioscapine . Brown ede pools , as wellde pools . TogetheBeyond the rational refactoring of plant natural product pathways for microbial biosynthesis of natural alkaloids, synthetic biologists are taking inspiration from medicinal chemistry campaigns investigating small-molecule drug leads, with the objective of expanding the repertoire of medicinal plant alkaloids by including their unnatural derivatives. Bioactive natural products often proceed from lead to licensing without undergoing significant modification . Howeverin vitro and in planta studies have characterized the turnover of halogenated intermediates and developed parts relevant to achieving this goal. These will be reviewed with respect to the pathway sections that have been refactored in yeast. Finally, how such strains would represent an unprecedented opportunity to develop semi-synthetic medicinal chemistry campaigns that probe entirely new regions of chemical space will be discussed  into natural product scaffolds, using the MIA and BIA pathways as test cases . Secondliscussed .Halogens form group seven of the periodic table and the biologically relevant members are fluorine, chlorine, bromine and iodine. Although the prevalence of these elements in nature is becoming increasingly understood, they are not often found naturally in plant alkaloids . ConversHowever, despite considerable interest and a number of engineering studies targeting individual enzymes , there hThe potential for enzymatic halogenation emerged with the discovery of widespread halogenation in nature, which pointed toward a rich trove of halogenating enzymes operating under benign conditions , 2018. TOf the numerous halogenases, the flavin-dependent halogenases (FDHs) have been a focus for biotechnologists, due to their high substrate- and regio-specificity, and their ability to function independently of carrier proteins that bind the substrate. A subgroup of FDHs targeting the indole moiety of tryptophan are the most well studied group of FDHs, due to the prevalence of indole as a building block in both biology and synthetic chemistry . MechaniOverall, there is a convincing body of evidence to suggest that halogenated derivatives of natural product scaffolds could be a rich source of improved or entirely novel pharmaceuticals. Furthermore, the site-specific introduction of halogens can facilitate production of a near-infinite number of other derivatives via cross-coupling reactions . While aThe well-documented effects of halogen atoms on the ligand binding and pharmacokinetic properties of pharmaceuticals present a tantalizing prospect of new or improved pharmaceuticals being created by introducing halogens into these classes of already bioactive compounds. This field remains in its infancy, but the number of studies reporting microbial synthesis of new-to-nature variants of medicinal plant alkaloids is growing. This section will review the progress made toward microbial synthesis of halogenated and other new-to-nature MIA and BIA variants, and describe protein engineering efforts predating microbial refactoring that can be integrated into emerging microbial strains. While this review will focus on MIAs and BIAs, the work performed to assess and engineer derivative turnover of these pathways provides an effective template that should be possible to emulate with other plant medicinal alkaloids .in vivo and in vitro, for the biosynthesis of new-to-nature derivatives of plant medicinal alkaloids  were designed to improve promiscuity for tryptamine derivatives, and the authors subsequently observed evidence of previously unobserved brominated MIAs  did not accept indole as a substrate. They therefore allowed the halogenases to target tryptophan and expressed an E. coli-derived tryptophanase (TnaA) to convert this into a halogenated indole, which could then be converted, with varying efficiencies, to chloro-indican in planta. As indole is a common biosynthetic precursor, and a privileged heterocyclic scaffold -norcoclaurine -reticuline. The authors took advantage of the improved yields of this strain to search for minor products indicative of NCS promiscuity, the enzymatic gateway to probing the derivative space surrounding the tetrahydroisoquinoline (THIQ) scaffold within norcoclaurine. By screening the liquid chromatography-mass spectrometry (LC-MS) spectra of supernatants for theoretical THIQ products, the authors identified peaks consistent with THIQs formed by the condensation of dopamine and endogenous yeast carbonyl species derived from L-phenylalanine, L-tryptophan and L-leucine  feeding of substrate analogs, (ii) pathway modification, and (iii) addition of enzymes, as recently reviewed by trans-acting aminoacyl-tRNA deacylase that selectively decouples fluoro-threonine from tRNA to prevent its integration into protein in Streptomyces cattleya (Beyond improving the catalytic activity of enzymes directly catalyzing in alkaloid production, several systems level challenges will also have to be overcome. Substrate toxicity will likely be a major hurdle on the way to producing some variants in yeast. For example, it has been shown that some tryptophan analogs interfere with tryptophan synthesis in yeast and inhibit growth , and maycattleya . Halogencattleya . AlthougEven as our understanding of how to engineer the promiscuity of these systems improves, the small number of successful studies on this topic have demonstrated the significant effort that is required to successfully produce new-to-nature variants. In addition to this, it is often difficult or expensive to source chemically synthesized intermediate analogs  and thisin vitro and in planta studies to be rapidly assessed. Although significant challenges still exist, if these challenges can be overcome, it is possible to envisage a new semi-synthetic approach to medicinal chemistry campaigns, specialized for diversifying natural product scaffolds. Such a system would involve microbial synthesis of medicinal alkaloid variants with halogen substitutions at specific points of the scaffold. These compounds can then be tested directly for pharmaceutical activities but also can be subjected to targeted, chemical cross-coupling reactions that can efficiently generate an almost infinite number of substituted variants. Thus, this system would combine natural mechanisms of generating diversity, i.e., using a small suite of organic building blocks combined through a large number of reactions, with synthetic mechanisms of generating diversity, i.e., using a wide range of building blocks combined with a small suite of reliable reactions (In summary, although still relatively few, the growing number of studies reporting yeast-based microbial synthesis of plant medicinal alkaloids are providing experimentally amenable contexts in which these pathways can be further engineered. This has major implications for furthering our understanding of new-to-nature variants of natural products but will also allow the combinatorial effects of the many previous learnings from eactions . As theseactions , we foreSB, JZ, and MJ conceived the scope of the review content and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.