Natural bone is a composite organic-inorganic material, containing hydroxyapatite (HAP) as an inorganic phase. In this review, applications of natural alginic acid (ALGH) polymer for the fabrication of composites containing HAP are described. ALGH is used as a biocompatible structure directing, capping and dispersing agent for the synthesis of HAP. Many advanced techniques for the fabrication of ALGH-HAP composites are attributed to the ability of ALGH to promote biomineralization. Gel-forming and film-forming properties of ALGH are key factors for the development of colloidal manufacturing techniques. Electrochemical fabrication techniques are based on strong ALGH adsorption on HAP, pH-dependent charge and solubility of ALGH. Functional properties of advanced composite ALGH-HAP films and coatings, scaffolds, biocements, gels and beads are described. The composites are loaded with other functional materials, such as antimicrobial agents, drugs, proteins and enzymes. Moreover, the composites provided a platform for their loading with cells for the fabrication of composites with enhanced properties for various biomedical applications. This review summarizes manufacturing strategies, mechanisms and outlines future trends in the development of functional biocomposites. At low pH, the precipitation of ALGH gel is observed. The gel-forming properties of ALGH provide a versatile platform for the development of advanced drug delivery technologies. ALGH can be crosslinked with metal ions or organic molecules for the fabrication of gels with enhanced mechanical properties, chemical stability, improved cell compatibility, surface wettability and desired morphology [Alginic acid (ALGH) is a natural biocompatible and biodegradable polymer, which is widely used for various biomedical applications. Low-cost natural alginates can be derived from brown marine algae. In physiological conditions, ALGH takes the form of a highly soluble sodium salt (ALGNa). ALGH is a polysaccharide, containing mannuronate and guluronate monomers . The anirphology ,3.2+ ions [ALGH is widely used for the fabrication of biomedical scaffolds for bone tissue engineering , capsule2+ ions .– and Na+:ALGH is used as a dispersing agent for the colloidal processing of materials . It is i− towards the anode, where the pH is low due to water decomposition and generation of H+ ions:The electric field provides the electrophoretic force moving ALG− is neutralized at the electrode by the positively charged H+ resulting in the ALGH film deposition:The negatively charged ALGThe development of EPD of ALGH films provided a platform for the fabrication of composite films containing proteins, enzymes and other functional materials in the matrix of ALGH ,24,25,26ALGH polymer has generated significant interest for the fabrication of composite materials, containing hydroxyapatite (HAP). It is important to note that natural bone is a composite organic-inorganic material . HAP is 2+ ions [As noted above, ALGH belongs to the family of anionic polysaccharide polymers. It is known  that pol2+ ions  and exhi2+ ions ,41,42. T2+ ions  and drug2+ ions .This review is focused on the manufacturing and functional properties of ALGH-HAP organic-inorganic composites. The goal of this review is to emphasize innovative ideas in ALGH-HAP biocomposite technology, which are based on the unique properties of ALGH polymer and HAP. The current state of the research field of ALGH-HAP organic-inorganic composites is reviewed and key applications are outlined. Recent advances in materials synthesis, design, colloidal and electrochemical deposition and surface modification methods, fabrication of composites and clinical performance are described, which have enriched the science and technology of ALGH-HAP composites.The literature data discussed above indicated that unique properties of ALGH polymer facilitated the development of ALGH films by different techniques, such as LBL, spin coating, dip coating and EPD. Such deposition techniques were further developed for the fabrication of ALGH-HAP films for advanced applications , The inc− species adsorbed on HAP particles and facilitated their charging and dispersion. The deposition mechanism involved electrophoresis of HAP nanoparticles, containing adsorbed ALG− toward the anode, where protonation of adsorbed ALG− in reaction (3) resulted in particle coagulation and deposition on the electrode surface. The deposition of composite involved EPD of ALGH and HAP, containing adsorbed ALGH. The film forming and binding properties of ALGH facilitated the formation of adherent and continuous ALGH-HAP films   and chlo greater . CHX is  greater .Investigations  revealedALGH-HAP scaffolds have been prepared in conjunction with several other polymers, such as gelatin (Gel) ,72,73, c− groups can electrostatically interact with the NH3+ groups of Chit to weakly bind them together. As well, the NH3+ groups can interact with the PO43- groups of HAP, binding the scaffold together and forming a more compact scaffold. The decrease in porosity and degradation rate and increase in mechanical stability due to these increased interactions in the scaffold were reported [The addition of Gel microspheres (mGel) to the scaffold further improved the mechanical properties of the scaffold. However, an excessive increase of mGel concentration decreases the mechanical properties due to its hydrophilic properties and the increased brittleness of the scaffold. The mGel were found to be homogeneously distributed throughout the scaffold walls. It slightly decreased the scaffold’s porosity by increasing the wall thickness. The incorporation of mGel increased the viscosity of the solution and, therefore, decreased the gelation time of the scaffold. The scaffold had a lower swelling capacity as the scaffold was more compact and less water could disperse into the scaffold . The increported . The gelreported . Chit-ALreported . ALGH coreported .Advanced strategies have emerged as convenient methods for the fabrication of scaffolds with desired microstructures ,87,88. ASignificant interest has been generated in the development of colloidal techniques, based on the use of ALGH as a dispersant for HAP . InvestiAnother promising strategy  is based2+ doped nano-HAP-ALGH-Gel were prepared for magnetic resonance imaging based on non-invasive monitoring of bone tissue regeneration [Among the interesting examples of scaffold applications is the development of ALGH based composite scaffolds, contained Sr-doped HAP. Such scaffolds showed enhanced compressive strength and promoted cell proliferation and osteoblast differentiation . Alginatneration . The incneration . ALGH-HAneration . ALGH-HAneration . Such scneration .Cell-based tissue engineering is an important platform for the development of advanced scaffolds ,106. It The use of dopamine modified ALGH has emerged  as an inALGH-HAP biocements have been developed as bone fillers for tissue engineering and orthopedics . BiocemeInvestigations highlighted the importance of powder to ALGH liquid ratio (P/L) for obtaining injectability and hardenability . The setVarious techniques have been developed for the fabrication of ALGH-HAP biocements ,97. FibrALGH-HAP biocements have been fabricated with the further addition of other materials, such as chitosan (Chit) ,123, gel2+ ions from CaCl2 solutions, which bind to the carboxylate groups of ALGH through ionic interactions, forming an “egg-box” pattern [ALGH-HAP gels have been developed for a wide variety of applications in tissue engineering and drug delivery . The amo pattern ,127, the pattern .HAP nanowires interacted with the gel through electrostatic interactions from hydrogen bonding. These nanowires are highly flexible, as they have a very small diameter and are long, which is beneficial for improving the mechanical properties, as compared to brittle bulk HAP. These gels can easily be molded into a wide variety of shapes and after the removal of the mold, the gel holds its shape well. The gels are highly elastic and have an increased tensile and compressive strength compared to pure ALGH gels. The ALGH-HAP gels have a much improved continuous porous structure compared to ALGH gels and the composite gel demonstrates a much higher density, similar to that of natural bone . The com2+ ions diffuse towards the bulk of the solution causing gelation as they diffuse. Addition of PO43– ions decreases the gelling velocity due to the consumption of Ca2+ ions during gelation and the formation of HAP [ALGH-HAP gels have been developed by the mineralization of HAP during the gelation process using brushite seed crystals. Can of HAP . This inn of HAP .3O+ ions are generated and their movement decreases the size of the gel due to electro-osmosis and electrophoresis [ALGH-HAP gels showed interesting stimuli-dependent properties that make them useful for drug delivery applications . Applyinphoresis . Gel swephoresis . Figure A variety of drugs, such as tetracycline ,130,131,ALGH-HAP gels have been developed incorporating other compounds such as collagen (Col) , gelatinALGH-HAP beads have been developed  for tissThe composite beads demonstrated a slightly roughened surface and nanoHAP was distributed homogeneously throughout the bead, which gives the bead a white color. Irregular, interconnected pores were found throughout the bead. These pores allowed the beads to retain large amounts of water, which allows the diffusion of nutrients throughout the bead for cell growth. The addition of nanoHAP is able to promote water diffusion into the bead due to the carboxyl and hydroxyl groups of ALGH, which can interact with the water molecules. The addition of nanoHAP increased the degree of crosslinking in the network by increasing the ionic strength. Due to this increase in crosslinking, the amount of swelling of the bead decreased with increasing nanoHAP content. This decrease in swelling further serves to restrict water and nutrient flow through the bead. The ALGH-HAP composite beads swell less in acidic media than basic media due to the protonation of the ALGH carboxylate groups in acidic media and the repulsion of the carboxylate groups in basic media. When the beads are placed in simulated body fluid, apatite mineral growth is seen on the surface of the beads. NanoHAP contains hydroxyl groups which serve as nucleation sites on the bead for this deposition . Figure − groups of the organic components and HAP further increase the degree of crosslinking in the bead and improve these properties [ALGH-HAP beads have been produced with the addition of a phenolic additive which reduced the degradation behavior of the beads in the presence of lysozyme, compared to ALGH beads and the addition of HAP further significantly reduced the degradation rate. They both also increase the mechanical properties of the beads. This modification strategy also serves to decrease the swelling ratio of the bead. These properties of the modified beads are due to the additional crosslinking providing more structural support, which serves to decrease the degradation rate and swelling. Chemical interactions between the functionaloperties .2+ crosslinking the matrix, which binds DS to the beads. Increasing ALGH concentrations allows for improved loading of DS in the beads. The beads have a cabbage-like pore structure, which forms barriers preventing drug release from the beads. To minimize drug burst release, the dissolution rate could be decreased and bead disintegration could be avoided. This can be achieved by increasing the HAP content, which minimizes the initial swelling and slows the crosslink breakdown [Multiple different drugs have been encapsulated in ALGH-HAP beads for drug delivery, such as diclofenac sodium (DS) , ibuprofreakdown .Ibu can be loaded into ALGH-HAP beads and Ibu binds to the beads through hydrogen bonding between the carboxyl groups of Ibu and the carboxyl groups of ALGH. The carboxyl groups of both Ibu and ALGH are deprotonated, which greatly increases the solubility of Ibu in the media, as well electrostatic repulsion between these deprotonated carboxyl groups leads to a fast release of the drug. However, the addition of HAP controls this release behavior in the same manner as DS. In acidic media, the low swelling restricts the release of the beads, as well the carboxyl groups of both Ibu and ALGH remain protonated; therefore, the hydrogen bonding further slows their release from the beads .The literature data described in this review indicated that ALGH based materials loaded with HAP are promising for bone tissue regeneration, treatment of bone defects, surface modification of implants, drug delivery, endodontics and other biomedical applications. The synergy of functional properties of ALGH and HAP offers a strong potential for the development of bone substitute materials with advanced microstructure and properties. The bioactivity of ALGH and HAP composites provides a platform for advanced implant applications. Composite materials have been developed in different forms, such as films, coatings, scaffolds, gels, beads and biocements. ALGH-HAP composites can be loaded with other functional materials for the fabrication of new composites with advanced functionality.New coating deposition mechanisms based on pH dependent charge and solubility of ALGH as well as ALGH adsorption on HAP are key factors for new emerging developments in the field of surface modification of biomedical implants. ALGH can be used as a biocompatible capping, dispersing and film forming agent for different film deposition techniques. Further advances can be achieved by the development of film crosslinking techniques to improve the chemical stability of the coatings. EPD offers the potential for co-deposition of ALGH and HAP with other functional biomaterials. Progress in this area can also be achieved by the optimization of ALGH molecular size, chemical modifications of ALGH molecules, optimization of the size and shape of HAP particles and their dispersion.Many examples throughout literature display the beneficial use of ALGH-HAP scaffolds for tissue engineering and drug delivery applications, combining the properties of the organic and inorganic components for superior scaffold properties. Different crosslinking methods are being researched to improve the mechanical properties and swelling of the scaffold. Much current research is focused on the 3D printing of such scaffolds, the optimization of bioink compositions, as well as the preparation of ALGH-HAP scaffolds combined with other polymers to augment the scaffold properties. New technologies for 3D printing of ALGH-HAP scaffolds facilitate the control of porosity, which is one of the key factors for successful biomedical applications. As such, many further advances can be made in these areas. Cell-based tissue engineering is a promising platform for the development of advanced scaffolds. It is promising to create living functional tissues for bone regeneration.ALGH-HAP biocements demonstrate excellent properties for bone filling applications in tissue engineering, with the possibility for local drug delivery in vivo. They display suitable cell growth and proliferation and setting times for clinical applications. The addition of other compounds to the ALGH-HAP cement can further modify the cement properties. Future research can develop cements with stronger mechanical properties for the potential use in load-bearing applications, as the mechanical strength of current cements is only suitable for non-load-bearing applications. Further advances can be achieved by the development of fully biodegradable cements to improve their use in tissue engineering applications.ALGH-HAP gels have many potential applications due to their moldability into a wide variety of shapes, their elasticity and ease of gelation in solution. The swelling of the gel is easily controlled by the degree of crosslinking of the gel and the pH of the media it is placed in. Much research is focused on the controlled release of drugs from these gels for local drug delivery. Further advances can be achieved through the optimization of the morphology of HAP dispersed in the gel.Significant interest has been generated for the use of ALGH-HAP beads in controlled delivery of different drugs. The porous structure that develops and low swelling is ideal for sustained drug release, minimizing the initial burst release. However, more research can further optimize the drug release properties for more controlled release.


Anaerobic glycolysis is the process by which glucose is broken down into pyruvate and lactate and is the primary metabolic pathway in sepsis. The pyruvate dehydrogenase complex (PDHC) is a multienzyme complex that serves as a critical hub in energy metabolism. Under aerobic conditions, pyruvate translocates to mitochondria, where it is oxidized into acetyl-CoA through the activation of PDHC, thereby accelerating aerobic oxidation. Both phosphorylation and acetylation affect PDHC activity and, consequently, the regulation of energy metabolism. The mechanisms underlying the protective effects of PDHC in sepsis involve the regulation on the balance of lactate, the release of inflammatory mediators, the remodeling of tricarboxylic acid (TCA) cycle, as well as on the improvement of lipid and energy metabolism. Therapeutic drugs that target PDHC activation for sepsis treatment include dichloroacetate, thiamine, amrinone, TNF-binding protein, and ciprofloxacin. In this review, we summarize the recent findings regarding the metabolic regulation of PDHC in sepsis and the therapies targeting PDHC for the treatment of this condition. Sepsis is a major healthcare concern caused by an aberrant response to infection, which is easily complicated with multiple organ dysfunction syndromes –3. DespiSepsis is characterized by the release of a large number of inflammatory mediators, the imbalance in energy metabolism and the accumulation of lactic acid. The mechanisms underlying sepsis pathogenesis and development are complex, and involve inflammation, immunity, and metabolism, among other processes. Recent studies have shown that in the early stages of sepsis, high level inflammation relies on anaerobic glycolysis as the key energy source, while in the recovery stage, inflammation level was lowered and the cells switch to fat oxidation for their energy supply .During the process of glycolysis, one molecule of glucose is broken down into two molecules of pyruvate. Under aerobic conditions, pyruvate translocates to mitochondria, where it is oxidized into acetyl-CoA through the activation of the pyruvate dehydrogenase complex (PDHC), thereby accelerating aerobic oxidation, whereas under hypoxic conditions, pyruvate produced through anaerobic glycolysis might result in the generation of lactic acid .via the former mechanism is significantly slower than that via the latter. Therefore, in the acute stage of sepsis, which is characterized by high energy consumption and hyper-inflammation, cells tend to generate energy to meet the significantly increased energy demands through anaerobic glycolysis, during which pyruvate converts into lactate. Furthermore, an absence of acetyl-CoA generated via pyruvate oxidation remodeled the tricarboxylic acid (TCA) cycle and lipid metabolism, with accumulation of two intermediates which could be produced through other pathways other than the physiological TCA cycle upon sepsis, namely succinate and citrate, both of which are pro-inflammatory mediators. In macrophages treated with lipopolysaccharide (LPS), succinate could be produced through anaplerosis of α-ketoglutarate into the TCA cycle and the subsequent increased glutamine metabolism. Moreover, LPS also increases γ-aminobutyric acid and its transporters, thus producing succinate. Citrate accumulates due to the shunting of cis-aconitate to itaconate through the enzymatic action of aconitase decarboxylase. Furthermore, free fatty acids and glycerol are released abundantly from adipose tissue into bloodstream, and are taken up by the liver to be oxidized into β-oxidation with the increase of acetyl-CoA, to produce more energy and ketone bodies, used as the source of energy by organs. However, this process is accompanied by the increased levels of palmitic acid and palmitoyl-carnitine, which might induce mitochondrial dysfunction. Together, lipotoxicity is also induced, characterized by the accumulation of malondialdehyde and 4-hydroxynonenal, which are the end products of lipid radical reactions and could cause cell death in liver and kidney  and promote the activation of nuclear factor-kappa B (NF-κB), as well as the further release of inflammatory mediators . StudiesHowever, switching between these two metabolic pathways is necessary and might have beneficial effects. It is reported that, during the early stages of sepsis, metabolism in tubular epithelial cells can switch from aerobic oxidation to anaerobic glycolysis, suggestive of metabolic reprogramming . This alConcerning the wide-ranging effects of metabolic reprogramming, both beneficial and detrimental, further work is warranted for an improved understanding of the role of the different metabolic pathways in sepsis, as well as identifying strategies to improve outcome in septic patients through the regulation of metabolism.A large number of studies have focused on PDHC, the key regulator of glucose metabolism, in exploring the underlying pathogenesis of sepsis . AccordiPDHC, a multienzyme complex composed of pyruvate dehydrogenase E1 (PDHE1), dihydrolipoamide transacetylase E2 (PDHE2), and dihydrolipoamide dehydrogenase E3 (PDHE3), serves as the critical hub in energy metabolism. Numerous studies have shown the positive regulatory role of PDHC in anaerobic glycolysis in sepsis. One study showed that PDHC activity was decreased by ~70% in skeletal muscle cells of septic rats, leading to cellular hypoxia and dysfunction . In termvia multiple mechanisms, including phosphorylation and acetylation. PDH kinase (PDK), consisting of four isoforms (PDK1–PDK4), acts as an upstream negative regulator of PDHC and is located in the mitochondrial matrix (via PDH phosphatase (PDP)-mediated dephosphorylation -6, IL-12, and IL-1β. This finding highlights that PDK inhibition exerts positive regulatory effects in glycolysis ediators . Bakalovediators . These rvia the mitochondrial citrate carrier (CIC), is a key molecule for the generation of energy. After citrate synthesis in the mitochondrial, citrate can enter the Krebs cycle and promote oxidative phosphorylation for energy production, which is required for the production of inflammatory molecules after LPS treatment. In the cytoplasm citrate is cleaved to acetyl-CoA which is the precursor for fatty acid, or cleaved to oxaloacetate which is converted to malate and then pyruvate  for 5 days in septic rats helped to induce the activation of PDHC in skeletal muscle and to significantly reduce the level of lactate, suggesting that amrinone may be useful for the treatment of hyperlactatemia in septic patients .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biomechanical forces play a major role in organ development, shape, and function. When exceeding the physiological range, however, they can become detrimental for organ structure and function. This is probably best exemplified by the cardiovascular system, with both the heart and blood vessels being continuously exposed to the biomechanical forces exerted by the pressure and flow of blood, which produce not only acute changes in cardiovascular function but also result in structural changes in the cardiovascular system.Cardiovascular Mechanobiology,” containing a total of 10 articles, with a balanced mix of 5 review and 5 original articles.The field of cardiovascular mechanobiology is a fascinating and clinically highly relevant Research Topic, with many research groups contributing to the advancement of our understanding of the acute and chronic adaptations and maladaptations produced by biomechanical forces. These insights continue to generate novel targets for therapy of a variety of cardiovascular diseases. Several aspects of cardiovascular mechanobiology are presented in this very timely Frontiers Research Topic “Jackson discusses the mechanobiology of myogenic tone in peripheral resistance arteries and arterioles, presenting the current understanding of the multiple mechanisms that have been implicated in myogenic tone, including the roles played by G-protein coupled receptors, ion channels, and protein kinases in vascular smooth muscle cells. The article highlights the areas where our knowledge and understanding of myogenic tone has remained incomplete and that warrant further research.In the first review article, Sporkova et al. review the mechanisms underlying the mechanobiological cues mediating the inward remodelling of small arteries and arterioles in response to chronic stretch produced by, for example, hypertension. Particular focus is on the role that LIM domain containing proteins play in mechanosensing and transduction in vascular smooth muscle and endothelial cells, translating extra- and intracellular mechanobiological cues into changes in gene expression in these cells that drive vascular remodelling.In the second review paper, Brandt et al. review the mechanobiological aspects of microvascular function and structure in health and disease with a particular focus on the coronary microcirculation. An in-depth review is provided on the factors involved in coronary microvascular functional and structural alterations in obstructive and non-obstructive coronary artery disease and the molecular mechanisms involved, providing novel potential targets for the treatment of ischemic heart disease.Islam et al. highlighting the emerging role of shear stress, cyclic strain, matrix stiffness, and composition in endothelial-to-mesenchymal transition, which is a hot topic particularly in maladaptive cardiac remodelling due to pressure overload but also in response to myocardial infarction. The authors also identify areas requiring further investigation.The mechanobiology of endothelial-to-mesenchymal transition and its role in cardiovascular disease is reviewed by Lityagina and Dobreva review novel insight into the epigenetic aspects of cardiovascular mechanobiology. Specifically, they describe how mechanical forces are transduced to chromatin through the tensed actomyosin cytoskeleton, the linker of nucleoskeleton and cytoskeleton (LINC) complex and the nuclear lamina, and the importance of these mechanisms in cardiovascular disease.Finally, Körner et al. tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-derived cardiomyocytes. The authors observed that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for excitation-contraction coupling. Moreover, soft substrates influenced contractile properties and improved intercellular coupling in iPSC-derived cardiomyocytes, indicating that mechanical stiffness of the cell environment drives iPSC-derived cardiomyocytes toward further maturation by inducing adaptive responses.In the original research articles various aspects of the mechanobiology of vascular smooth muscle cells, endothelial cells, as well as cardiomyocytes were investigated. Using inducible pluripotent stem cell (iPSC)-derived cardiomyocytes, Akinbote et al. investigated vessel-stroma crosstalk in normal conditions and in the presence of fibrosis, using human iPSC-derived endothelial cells co-cultured in the absence and presence of primary human cardiac and lung fibroblasts in a microfluidic device to generate cardiac and pulmonary-like microvasculature that can be perfused at near physiological flow rates. Their study not only demonstrates the strong impact of stromal-endothelial cell interactions on vessel formation and extravascular matrix regulation, but their human 3D in vitro set-up could be very useful in future studies examining anti-fibrotic therapies on patient-specific iPSCs.In the next article, Morel et al. investigated the effects of low and supra-high aneurysmal wall shear stress on porcine arterial endothelial cells. The authors observed that differential regulation of gene expression observed under various wall shear stress conditions translates into a different organisation of the endothelial cell architecture, suggesting that this adaptation of endothelial cells to different aneurysmal wall shear stress conditions may affect vascular remodelling in intracranial aneurysms.Wall shear stress has been proposed to influence intracranial aneurysmal growth and rupture. Consequently, Liu, Bankell et al. investigated the role of alterations in MRTF signalling in vascular inflammation, and observed a cell type specific  suppression of inflammatory mediators. In a second study, Liu, Rippe et al. studied the regulation of muscarinic receptors by MRTFs and observed that muscarinic receptor M3 expression was driven by MRTFs in a cell specific manner, i.e., by MRTF-B/SRF in endothelial cells and by myocardin/SRF in vascular smooth muscle cells.Myocardin related transcription factors (MRTFs), including myocardin itself, MRTF-A, and MRTF-B are co-factors of serum response factor (SRF) that activate the smooth muscle cell gene program and play a key role in smooth muscle cell differentiation and mechanobiology. These highly diverse review and original research articles, covering a wide range of studies, illustrate the broad biological implications of mechanosensing and mechanotransduction in the cardiovascular system. We wish you an interesting and enjoyable journey through this Frontiers Research Topic!All authors contributed equally to the writing of this article and organisation of the Research Topic.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD). AAT, the major serine protease inhibitor (Pi), is encoded by two codominant independent alleles. Heterozygous individuals (Pi MZ) synthesize two pools of the protein, the normal M fraction is entirely exported, while 85% of the abnormal Z is retained in the liver. Under conditions of clinical stimulation, the Pi MZ phenotype undergoes a unique phenomenon, characterized by an increase of synthesis of both fractions, but a divergent behavior in export. Moreover, 100% of the M fraction, but only 15% of the Z, are secreted into the blood. That results in an elevation of serum AAT levels up to the normal range associated with an increase of the amount of hepatic storage. The phenomenon has been called “Recruitment-Secretory Block” (“R-SB”) and explains why heterozygous Pi MZ individuals are not prone to develop the extrahepatic manifestations observed in Pi ZZ homozygotes as a consequence of the severe serum AAT deficiency.HHHS occurs only in the heterozygous state. The “R-SB” phenomenon neither apply to homozygous Pi ZZ nor to heterozygous HHHS individuals, thus suggesting that the homozygosity of HHHS could be incompatible with life.In contrast to fibrinogen that is synthesized exclusively and selectively by the liver, AAT can be synthesized by various tissues and cell types, a.o. macrophages, bile duct, pancreatic islet and sperm cells. Simultaneous accumulation of AAT in the RER of both hepatocytes and these cell types, indicates a mutation and a primary synthesis. Cells other than hepatocytes do not respond to acute phase stimuli and do not contribute to the circulating levels.Indeed, Pi ZZ cirrhotic patients, after liver transplantation from a Pi MM donor, acquire permanently the donor’s phenotype and correct the serum AAT deficiency. Based on morphological, physiological, and serological correlations, it is suggested that AAT in cells other than hepatocyte is packaged in condensing vacuoles, lysosomes, and endocrine granules in the form of protease–antiprotease complexes and is playing an intracellular role.The liver is the only source of fibrinogen. Therefore, the mutations causing conformational molecule abnormalities, result in exclusive hepatic storage.The storage of mutant AAT and fibrinogen is toxic for the hepatocytes and causes chronic liver disease and cirrhosis.Molecular analyses of the storing mutations and 3D modeling studies have localized the mutations in critical sites for correct assembly and secretion and have clarified the pathomorphogenesis of the aggregation process of the proteins within the RER.In this article, we review clinical and experimental published data collected over 40 years. Taken together, the results appear to be of potential utility with regard to therapeutic strategies aimed to cure ERSD. In particular, they discourage the use of drugs that would increase the synthesis without a simultaneous increase of secretion. This article also addresses future research directed at small molecules that can prevent intracellular accumulation and increase the degradation of the mutant proteins.Endoplasmic reticulum storage diseases (ERSD) are genetic disorders affecting secretory proteins. Due to mutations in the encoding genes, the proteins may acquire an abnormal conformation of the molecule that aggregates within the rough endoplasmic reticulum (RER) instead of being regularly exported . The proThe prototype of ERSD is alpha-1-antitrypsin deficiency (AATD). Its discovery represents a milestone in the medical field as it has led to clarify the pathogenesis of a subset of liver cirrhosis previously considered cryptogenic . It has The original recognition of AATD patients was done on the basis of identification on serum isoelectric focusing (IF) on polyacrylamide gel  of a varThe normal variant is called M and is present in about 88% of the general population , and twoPi S  and Pi NAAT is expressed in several tissues and cell types. However, the role, as well as the function of the protein in cells other than hepatocytes, need to be clarified.The identification of the first mutation in the fibrinogen gamma chain gene  and the This review is an update of the immunomorphological, electron microscopic (EM), and molecular studies on AATD and HHHS. The rationale was based upon the assumption that finding a secretory protein stored in the RER of a given cell is a simultaneous indication of an underlying mutation and of a primary synthesis of the protein.In this paper, we revised all material from our previously published clinical and experimental work carried out over the past forty years. Material, methods, patients, and ethical approval had been reported in details in the pertinent articles cited in the References.Before reviewing the pathophysiology of the causative mutations and the morphological hallmark for diagnosis, we will first introduce some aspects of the synthesis and function of the two proteins.In addition, we will discuss the relationship between the intracellular and extracellular AAT and fibrinogen in normal conditions and in ERSD, in view of their potential towards future strategies finalized to the treatment of ERSD.According to the purposes of this review, we will summarize and discuss published data from the literature related to the variants of AAT where serum deficiency is due to the accumulation of the mutant protein variants  in the liver. The hepatocytic storage of mutant AAT presents in the form of PAS diastase (PAS–D) resistant inclusions immunoreactive to anti-AAT specific antibodies, corresponding to material in the RER a–c.The molecular mechanism of Z AAT accumulation in the liver has been demonstrated as due to a loop–sheet polymerization following a molecular interaction between the reactive center loop of one molecule and the gap in the A-sheet of another . The disAAT is an acute phase reactant protein and the major inhibitor of serine protease (Pi) ,18. In aThe circulating AAT is synthesized by hepatocytes, exported into the blood ,24,25,26Three cell types in the liver are capable of AAT synthesis: (i) hepatocytes, (ii) biliary, and (iii) Kupffer cells.(i) Hepatocytes synthesize AAT like all secretory proteins. The AAT mRNA transcribe the message on polysomes attached to the granular endoplasmic reticulum (RER) that translates, synthesizes, and discharges the polypeptide chain into the lumen of the RER. The first sugar attachment (“core glycosylation”) occurs during translation. Then AAT translocates from the RER to the SER where further glycosylation, trimming, and elongation occur, and reaches the Golgi apparatus where terminal glycosylation takes place. Finally, the mature protein is exported into the circulation with the cooperation of microtubules ,24,25,26In case of storing mutations , AAT polymers aggregate within the RER cisternae and are visualized under the transmission EM c.AAT is encoded by two codominant independent alleles, which can occur in various combinations. The normal homozygous phenotype is designated Pi MM. The abnormal Z allele can present in a homozygous (Pi ZZ) or heterozygous (Pi MZ) state, corresponding respectively to severe or partial (intermediate) AATD. Each allele is responsible for the synthesis of 50% of the entire pool. Under conditions of clinical stimulation, Pi MZ individuals are capable of raising serum AAT levels by three–four fold, as the M fraction, due to the acute phase reactant nature of the protein, undergoes an increased synthesis and secretion rate, while 85% of the Z fraction is retained within the cell and contributes to increasing the amounts of intracellular storage. This reaction of Pi MZ to acute phase stimuli has been designated Recruitment-Secretory Block (“R-SB”) phenomenon . In Pi ZThese data, together with the clinical observation that AATD (Pi ZZ) cirrhotic patients, after liver transplantation from a Pi MM donor, acquire the phenotype of the donor and normalize the serum levels , have de(ii) Biliary cells (Hering and bile duct cells) synthesize and export AAT into the biliary system and contribute to the bile AAT pool that is estimated to represent 30% of the circulating AAT . The capThe exact amount of AAT exported by biliary cells in not known. The bile AAT is mostly derived from transepithelial bypass of bile duct cells in human and hepatocytes in rats. Circulating dimeric IgAs as well as those derived from mucosal plasma cells, contain three ligands: J chain linking two IgA molecules, secretory component (SC) and one available to bind AAT. The SC recognizes a receptor on the basolateral membrane of epithelial cells. Subsequently, the complex (IgA/AAT/IgA/SC/J chain) is internalized via endocytosis and, through a vesicular mediated system, discharged into the lumen of bile ductules and ducts .AAT bound to IgA maintains its anti-protease activity and protects the immunoglobulins from proteolysis .IgA and AAT play an important role in neutralizing, within the biliary system and intestine , bacteri(iii) The third cell types capable of AAT synthesis in the liver are Kupffer cells. Kupffer cells are sinusoidal lining cells belonging to the monocyte/macrophage system. Like all macrophages, Kupffer cells express AAT either in normal Pi M a or in PIn Kupffer cells from Pi Z specimens, AAT-like material has been detected in the lumen of the RER and of the channel network of the SER, thus proving a primary synthesis and capacity of translocation in these cells .However, Kupffer cells do not contribute to the circulating AAT. Therefore its presence in the ER but not in trans-Golgi SV suggests a different meaning than the hepatocytic one.On the basis of the knowledge on protein synthesis and morphological observations, AAT could be synthesized in the RER and reach the lysosomes via the Golgi Endoplasmic Reticulum Lysosome (GERL) area, according to the “addressing organelle signal” inserted early during translation of secretory proteins ,24,25,26AAT immunoreactivity has been demonstrated in monocytes/macrophages ,36 polymImmunoreactivity however, per se, is not proof of primary synthesis. In this study we have assumed that finding a mutant protein within the RER of a given cell, is a proof of primary synthesis in analogy with the presence of a given protein in a monolayer medium-free cell culture, although the final proof would be the demonstration of functioning mRNA in that cell.Gene expression of AAT has been demonstrated in human monocytes/macrophages in both Pi MM normal phenotypes and AATD ,44. TherOur EM observations have shown that Kupffer cells have not only the capability of AAT primary synthesis but also of translocating the protein to the SER. As Kupffer cells are not exporting AAT into the circulation, one can reasonably assume that they use the protein for intracellular purposes. At the level of the GERL area, AAT would recognize an endomembrane specific receptor and would be diverted to lysosomes, according to the organelle addressing signal incorporated at the time of the early synthesis ,24. InsiAAT in polymorphonuclear leukocytes, mast cells, Paneth cells containing large amounts of proteolytic enzymes, fulfills intracellular requirements, namely protection against proteolytic self-digestion, and discharge the uncoupled P and Pi into the extracellular/interstitial matrix, after Pi cleavage.The same pathway and function could be shared by endocrine cells. Once incorporated into endocrine granules  at the GERL level, AAT would temporarily form Pi/P complexes and inhibit the proteases that would activate pro-hormones into hormones, after AAT cleavage  45]..45].To synthesize, the consistently AAT positive staining in islet and paracrine pancreas  and in KOn the other hand, the consistently M AAT positive staining in islet and paracrine pancreas  and in KIn this article, we provided new findings on the neglected role of AAT in cells other than hepatocytes. The direct transfer of morphological findings into physiological or pathological interpretations, requires always caution. That holds true especially if the observations are new. A topical example is the different behavior of AAT in macrophages of malakoplakia and Whipple’s disease, two diseases of the monocyte/macrophage system that apparently share the pathomorphogenesis. Malakoplakia is a granulomatous histiocytic lesion mainly occurring in the urinary tract, resulting from a massive proliferation of von Hansemann macrophages . The cytE. coli. These findings concur to point out a lysosomal loss of function possibly due to the lack of P/Pi complexes dissociation ..99].Under the EM, the stored fibrinogen presents as densely packed tubular structures arranged in curved bundles (fingerprint like) or in parallel metameric arrays (fiber glass like)  12,13,1,15,16. ILike in AATD, the storage of fibrinogen in HHHS represents the elementary lesion of the liver pathology and is invariably associated with liver cell necrosis, hepatitis, fibrosis, and cirrhosis, mostly in early childhood.The mechanism of liver damage in HHHS is poorly understood. In analogy with AATD, one may argue that the cell engorgement and the over-distension of RER cisternae could interfere with other organelles and cell function.So far, in contrast to AATD, no single HHHS patient has developed HCC. However, it is important to underline that any poorly differentiated tumor, either in primary or metastatic sites, showing immunohistochemical positivity for fibrinogen can be diagnosed as HCC ,67, due The discovery of HHHS has allowed to identify the cause of a number of cases of liver cirrhosis that were previously considered cryptogenic, especially in children.The new disease has been first described by pathologists in liver tissue specimens from patients with signs of chronic liver disease, unsuspected for suffering from fibrinogen abnormalities . InitialIn that respect, indeed, the first case report is paradigmatic. The patient was a 64-year-old female undergoing surgical cholecystectomy, a duodenal polypectomy and a surgical wedge biopsy because of unexpected liver cirrhosis . After tSince then, seven more mutations on fibrinogen gamma chain with similar features have been found over the world . In addiAn intriguing phenomenon has been repeatedly reported with a few A-alpha chain mutations that result in systemic and especially renal amyloidosis ,106. ThiAAT and fibrinogen can undergo molecule conformational abnormalities as a consequence of gene mutations and accumulate within the RER of hepatocytes. These features characterize ERSD  and confIn addition, the two related diseases, AATD and HHHS, share cytotoxic liver cell damage and cirrhosis due to the storage. The latter is the hallmark of either diseases and represents the elementary lesion thus strengthening the genotype-phenotype correlation ,58. LiveInterestingly, AATD and HHHS delineate a link between intracellular and extracellular conformational diseases and between liver and kidney with regard to renal amyloidosis that is cured by replacing the patient’s liver which, despite its normal structure and function, indirectly causes the renal pathology by producing an abnormal A alpha fibrinogen chain.There are however several differences between the two proteins and the two diseases. In contrast to fibrinogen, AAT can be synthesized by several cell lines and tissues: macrophages, polymorphonuclear leukocytes, mast cells, Paneth cells, pancreatic islet, and sperm cells. As none of these cells contribute to the circulating AAT levels, it is argued that AAT in those cells plays a different role than the circulating one. This means that while AAT remains a preeminently secretory protein, it can be localized in lysosomes, condensing vacuoles, endocrine granules, and plasma membrane in cells other than hepatocytes. This intracellular AAT can be synthesized in the RER and proceed along the secretory pathway to the GERL area where it is diverted towards organelles, or it can be synthesized by free ribosomes with an addressing signal inserted during translation.The intracellular AAT maintains the antiprotease activity and, within the cells, is presumably bound to proteases. The P-Pi complex would protect cell and organelles from self-digestion. The cleavage of AAT would allow the activation of pro-hormones into hormones as well as of proteolytic enzymes such as acrosin thus priming sperm cells to penetrate the zona pellucida of ovum.The reason way in AATD, all hepatocytes can show accumulation of the mutant protein, while other cells appear to undergo storage rarely, is explained by the different role played by the protein. In the liver, AAT is per excellence an acute phase reactant protein and, as such, is recruited for a major synthesis and export to raise the serum level, whilst in other cell types which do not contribute to the circulating level, AAT plays an intracellular role and its synthesis takes place at a steady-state rate.In conclusion, this paper has reviewed the results from original experimental studies for a Ph.D. thesis , and furThe protean behavior of AAT is granting AAT as “a protein for all seasons”. Further studies are needed to demonstrate AAT mRNA in extrahepatic locations and to confirm the relationship between intra and extracellular protease inhibitor activity.The striking similarity of intrahepatocytic aggregated fibrinogen and extrahepatic polymerized fibrin as well as the lack of overt hematological manifestations despite the very low fibrinogen level in HHHS, represent a challenge. The presumptive incompatibility with life of homozygous fibrinogen gamma chain mutations causing HHHS ,94 requiUseless and harmful could be the use of drugs, such as hormones or anabolizing steroids , as for Authors Contribution: F.C. conducted the study design and wrote the manuscript; I.G. and P.F. performed the immunohistochemical and molecular analysis studies. All authors have read and agreed to the published version of the manuscript.


Medicaid financing of nursing home (NH) care provides the strongest safety net for low income older adults, persons who have high-intensity long-term care (LTC) needs, and consumers with exorbitant LTC costs. Yet, NHs currently face serious threats to their financial viability, particularly in the context of the COVID-19 pandemic, where the costs of caring for residents in a safe way have increased significantly, even as the ability to recoup these costs from the Medicaid program has been constrained. The purpose of this study is to assess key demand and supply factors affecting the performance of the NH industry in Pennsylvania over time. It draws from several large, national data sources, including NH Compare, LTCFocus.org, the U.S. Bureaus of the Census and Labor Statistics, and Certification and Survey Provider Enhanced Reports, as well as state-level population projections and Departments of Health and Human Services data. An aggregate database was constructed with historical data points at the facility, regional, and state level. Annual total and regional trends were examined from 2010 to 2020. Findings suggest a growing gap between what NHs require to meet the needs of residents and the level of reimbursement paid by the largest funder: Medicaid. Considering demographic trends, this gap will only grow over time in the absence of policy change. The pandemic has further highlighted the existing challenges resulting from an underfunded service infrastructure and the need for additional investment if NHs are to provide high quality care to a growing cohort of older adults requiring support.


The present study aimed to demonstrate the possibility to treat corneal neovascularization using the combination of anti-VEGF injection and argon laser photocoagulation. Since the first antiangiogenic drug (AGF) launched in 2004, called Macugen (pagaptamid), Baush and Lomb and others have succeeded in promising efficiency, all aimed at treating Age-Related Macular Degeneration (AMD). We referred to the following: Avastin (bevacizumab) Roche; Lucentis (ranibizumab) Novartis; Eylia (aflibercept) Bayer. Later we started to observe that its application would solve cases of neovascularization in the anterior segment, such as rubeosis iridis, etc.The purpose of this report was to show that the injection of AGF in association with the argon laser could inhibit corneal neovascularization secondary to some inflammatory process.1].Among its various functions, the cornea plays a fundamental role in the transmission and refraction of light that falls on the eyes. For an adequate process of light transmission to the photoreceptors of the retina, the cornea must be transparent, aiming at the formation of the image [2]. This “privilege” consists of a balance between inhibitory and stimulating factors for angiogenesis in the corneal epithelium; and an imbalance in this process, caused by an inappropriate local tissue reaction secondary to several factors, including infections, traumas, and immunological reactions, can induce corneal neovascularization (NVK) [3]. Neovessels present structural changes such as the absence of pericytes and basement membrane, which allow fluid, proteins, and lipids to leak into the extracellular space. This culminates in edema and infiltrates, which reduce the transparency of the cornea and, consequently, visual acuity. In addition, neovessels favor the rejection of corneal transplantation due to the easy access of antigen-presenting cells to donor tissue [4]. NVK is the leading cause of corneal blindness in the world (trachoma) and in industrialized countries (herpetic keratitis) [5].This process of corneal transparency is called “Corneal angiogenic privilege (lymph)”, and, in the absence of pathology, it can inhibit the formation of blood and lymph vessels, which can cause corneal opacity and prevent the physiological process of passing light rays . In addition to treating the underlying cause, the treatment of NVK involves the destruction of the new vessels and restoration of the zone of angiogenic privilege, preventing a new neovascular process. Therefore, the approaches can be classified into three categories: anti-angiogenic, angio-regressive, and angio-occlusive [7].The choice of appropriate treatment for NVK can be challenging, and several models have been tested for the various models of NVK [9]. These compounds have also been shown to be effective in inhibiting lymphangiogenesis, both from the direct action on the lymphatic epithelium, as well as indirect on the release of macrophages, which is a promise in their use to inhibit graft rejection [7]. Despite the antiangiogenic action, anti-VEGF can cause adversities such as delay in endothelial recovery and decreased corneal thickness due to the neurotrophic effect of VEGF [10]. In this sense, studies are still needed to clarify drug efficacy, dosage, and safety.Anti-angiogenic agents (anti-VEGF) can be administered by topical ocular, subconjunctival, intrastromal, and intravitreal agents. Bevacizumab  is a recombinant humanized monoclonal antibody that recognizes all forms of VEGF and inhibits the VEGF/ VEGF-R interaction. It was initially approved by the FDA for the treatment of metastatic colorectal carcinoma but is effective in AMD and several studies have corroborated its usefulness in NVK. Ranibizumab  is produced from the Fab fragment of the same antibody used to produce bevacizumab and binds more strongly to VEGF-A [4]. PAL is a safe method, but it can cause complications related to inflammatory overload, in addition to causing iris atrophy, pupil ectasia, and hemorrhages [8].Photocoagulation (FCG) with argon laser is an angio-occlusive component, usually applied in association with bevacizumab; studies show that the drug’s effect is greater when simultaneously applied to chemical cauterization [The aim of the article was to evaluate the effect of AVEGF associated with PAL in the reduction of corneal neovascularization. The decrease in neovessels in the cornea proved that the combination influences the improvement of the transparency of the cornea and, consequently, the visual acuity; thus, improving the prognosis and a possible corneal transplant.Conflict of interestThe authors declare no conflicts of interest.Informed Consent and Human and Animal Rights statementInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of Ciências Médicas College, Belo Horizonte/MG, Brazil.AcknowledgementsNone.Sources of fundingThe authors declare that their research has not been funded by any entity. All authors had no financial disclosures or support in this work.DisclosuresNone.


Baclofen withdrawal syndrome represents a clinical emergency that can lead to life-threatening complications. It is often a diagnostic challenge because of its nonspecific nature of presentation and degree of symptom overlap with other clinical diseases. Electroencephalography (EEG) might provide important supporting evidence when neurological complications are involved. We present the case of a 55-year-old woman with sudden onset of motor manifestations at the limbs and an altered mental status 24 hours after cessation of intrathecal baclofen administration, following the removal of the pump due to infection, in whom a computed tomography did not show any acute-onset brain injuries, and multiple EEG recordings were performed. The first EEG showed the presence of bilateral sharply contoured waves, in the absence of epileptic discharges and seizures. No correlation between motor manifestations and EEG changes were detected. This EEG pattern was considered to be the expression of an overexcitation of the central nervous system (CNS) due to the loss of baclofen inhibitory effects, excluding an epileptic origin of motor manifestations. Another EEG, performed 24 hours later, showed the presence of triphasic waves with severe generalised slowing, suggesting the presence of encephalopathy. The last EEG, performed 48 hours after the previous recording, when a recovered state of consciousness was already present, showed regression of the triphasic waves and a reorganisation of the background activity. In our case, repeated EEG evaluation allowed monitoring the evolution of acute encephalopathy developed during baclofen withdrawal syndrome, from the initial phase of CNS hyperexcitability, through the phase of metabolic encephalopathy, and to its resolution. This modality allowed for optimising the diagnostic-therapeutic management of the patient during her stay in the intensive care unit. Baclofen, an agonist for gamma-aminobutyric acid (GABA) receptors , is an eWe present the case of a patient with sudden onset of anxiety and nonspecific motor manifestations at both the lower and upper limbs 24 hours after cessation of intrathecal baclofen administration, following the removal of the pump due to infection, in whom multiple EEG recordings were performed over time and contributed to proper clinical workout of the patient.μg/day. She showed normal liver and kidney function markers. She was admitted to an inpatient ward of Careggi Teaching Hospital on 29 October 2021, for the removal of the baclofen pump due to infection. Twenty-four hours after cessation of baclofen administration, the patient showed a sudden onset of anxiety followed by nonspecific motor manifestations at the lower and upper limbs. Therefore, she was started on oral baclofen (25 mg twice a day) alongside the benzodiazepines (diazepam and clonazepam), with only a partial reduction of motor manifestations which were still present at the lower limbs. She underwent brain computed tomography that did not show acute-onset brain injuries even after the administration of contrast medium. The patient was thus admitted to the ICU for proper diagnostic and therapeutic management, undergoing vital sign monitoring, oral administration of baclofen (25 mg three times a day), and intravenous administration of midazolam (0.02-0.05 mg/kg/hour), propofol (1-2 mg/kg/hour), and dexmedetomidina (0.15 μg/kg/hour), however without intubation. A blood gas analysis at admission to the ICU showed a partial pressure of oxygen (pO2) of 93 mmHg, a partial pressure of carbon dioxide (pCO2) of 38 mmHg, and pH values in the range of normality. A hemodynamic stability was detected during all the ICU stay.A 55-year-old woman with paraplegia, following a spinal cord injury at D4 level, from dorsal meningioma surgery in 2013, started baclofen intrathecal treatment for spasticity in 2017 with a dose of 75 Based on suspicion of an epileptic origin of the motor manifestations, the patient underwent an EEG about 4 hours after cessation of midazolam administration and 1 hour after cessation of propofol administration. The EEG pattern showed the presence of abundant and high sharply contoured bilateral waves  in the aTwenty-four hours later, another EEG was performed some hours after the cessation of intravenous anaesthetic drug administration. The EEG pattern showed the presence of abundant triphasic waves with severe generalised slowing, suggesting the presence of encephalopathy. The topography of the background activity was no longer detected . ClinicaThe recent expansion in the use of baclofen to treat spasticity of different causes has been associated with a significant increase in its complications, including toxicity events and withdrawal syndrome, especially regarding intrathecal baclofen administration . In partThis neurological manifestation may develop as a result of several clinical conditions occurring during baclofen withdrawal syndrome, including elevation in creatinine or in potassium levels and culminating in severe multisystem organ failure. The use of antiepileptic drugs, especially when excessive, might worsen the metabolic encephalopathy and, consequently, exacerbate the altered mental status associated with this clinical condition. Performing EEG could thus provide two advantages: on the one hand, it allows clinicians to distinguish between an epileptic or a nonepileptic origin of any patient's motor manifestations during the course of withdrawal syndrome, allowing improvement of the therapeutic management. On the other hand, it might allow clinicians to identify the possible development of metabolic encephalopathy.In our case, the first EEG, performed at an early stage after the onset of the motor manifestations of the limbs, allowed us to determine a nonepileptic origin of the clinical signs, thus avoiding starting inappropriate antiepileptic therapy. Indeed, the presence of the abundant high sharply contoured waves, in the absence of epileptic discharges and seizures, could be considered the expression of CNS overexcitation due to the loss of the inhibitory effects of baclofen.Avoiding the administration of inappropriate antiepileptic treatment was crucial in our patient, in whom encephalopathy developed the day after the onset of the first neurological symptoms. In the present case, in which withdrawal syndrome occurred because the pump had been removed due to infection, the second EEG pattern characterised by severe generalised slowing and abundant triphasic waves, signs of encephalopathy, could not distinguish between a septic or a metabolic origin of the neurological manifestation. However, the prompt (<48 hours) resolution of the parossistic triphasic discharges on EEG, which also showed reorganisation of the topography and only minimal slowing of the frequency of the background activity, in the absence of fever and elevated biochemical markers of sepsis (such as increased white blood cells) guided us to determine the encephalopathy had a metabolic origin related to baclofen withdrawal syndrome, rather than a septic cause.Withdrawal syndrome represents a concerning complication of baclofen therapy. Prompt recognition and great clinical suspicion are mandatory because if not treated, withdrawal syndrome might lead to multisystem organ failure and even death within few days. In our case, baclofen withdrawal syndrome therapy comprised reinitiation of baclofen and the use of drugs that mimic the mechanism of action of baclofen, such as benzodiazepines and propofol, besides hydration to prevent and treat rhabdomyolysis, vital sign monitoring, and cardiopulmonary support , 12. In


Zheng Jia in traditional Chinese medicine (TCM) books. Guizhi Fuling Pills (GFPs), a classic prescription for promoting blood circulation and removing blood stasis, is widely used for women's blood stasis diseases represented by Zheng Jia. At present, it has been applied to treat EMs in clinical settings. In this review, we systematically summarized the active ingredients and pharmacological mechanism of five Chinese herbs contained in GFPs and clinical applications of GFPs. The potential pathways of GFPs in the treatment of EMs were explored through network pharmacology. The current researches results indicate that the mechanisms of GFPs in the treatment of EMs mainly include acesodyne, anti-inflammation and improvement of hemodynamics. The main active compounds that are responsible for pharmacological effects in five Chinese herbs are paeonol, pachymic acid, cinnamaldehyde, amygdaloside and Paeoniflorin. This review can lay the foundation and identify the research direction for the development of GFPs as a new drug therapy for the treatment of EMs.Endometriosis (EMs) is recorded as  Endometriosis (EMs) is defined as the presence and growth of endometrial-like tissue outside the uterus, which is mainly characterized by dysmenorrhea, dyspareunia, pelvic pain and infertility Synopsis of the Golden Chamber written by Zhang Zhongjing in the Eastern Han Dynasty, the prevention and treatment of women's pregnancy diseases, postpartum diseases and other common diseases had been systematically recorded. Moreover, the book summarized a wealth of treatment experience and medication principles on gynecology diseases. Classic famous prescription GFPs is a representative TCM pill in this book. Its efficacy is to promote blood circulation, remove blood stasis, eliminate abdominal mass and stop vaginal bleeding. Recently, it has been mainly used in the clinical treatment of EMs as well as adenomyosis (AM) TCM pills are one of the main forms of traditional Chinese medicine preparations, which are mostly applied in the clinical treatment of chronic diseases and regulation of body function Zheng Jia in TCM, which was first recorded in Huangdi Neijing. Chinese medicine scholars named all the lumps in the human body as Zheng JiaSynopsis of the Golden Chamber had discussed in detail that lower abdominal pain, abdominal mass and irregular vaginal bleeding were the main clinical manifestations of Zheng Jia. Thus, Zheng Jia, as a gynecological disease, usually refers to the agglomeration in female lower abdomen accompanied by abdominal distension, pain, vaginal bleeding and other symptoms. AM, EMs, polycystic ovarian syndrome (PCOS) and reproductive system tumors all can be regarded as Zheng Jia according to its symptoms and pathological signs Zheng Jia is also similar to the EMs in TCM. TCM theory explains that the pathogenesis of Zheng Jia is mainly related to blood accumulation and qi stagnation in the uterus, which form lumps over time. TCM proposes that EMs is caused by retrograde menstruation, which results in blood stasis blocking the uterus for a long time. Thus, blood stasis is the core pathogenesis and pathological essence of both Zheng Jia and EMs.There is no relevant record about EMs in TCM books. According to its clinical manifestations, it can be considered as Zheng Jia, the relevant therapeutic methods are suggested in the Synopsis of the Golden Chamber “Chapter 20, the pluse, syndrome and treatment of women during pregnancy”. Promoting blood circulation, removing blood stasis, warming and dredging meridians are the core therapeutic methods. Subsequently, the classic prescription GFPs is established. The prescription is composed of 5 traditional Chinese herbs: Cassia Twig (Cinnamomum cassia Presl), Poria Cocos (Poria cocos(Schw.)Wolf), Peach Kernel (Prunus persica (L.) Batsch), Red Peony Root (Paeonia veitchii Lynch) and Cortex Moutan (Paeonia suffruticosa Andr.). Poria Cocos plays a role in infiltrating dampness and strengthening spleen. Peach Kernel and Red Paeony root have effects on promoting blood circulation and relieveing pain. Cortex Moutan is commonly used to clearing the heat caused by blood stasis Based on the pathogenesis of Shen Nong's herbal Classic and was commonly used in clinic. CT has the efficacy of sweating and slackening muscles, warming and dredging meridians, and assisting yang and transforming qi el. indicated that cinnamaldehyde could reduce inflammatory reaction by activating NF-κB signal pathway and reducing the production of interleukin-1 beta (IL-1β) and TNF-α el. confirmed that volatile oil of CT could reduce the expression of VEGF, COX-2 and other factors in ovarian tissue of rats, which could effectively treat infertility CT which is the dried twig of Cinnamon was first recorded in the el. proposed that p38MAPK which was phosphorylated by IL-1 was expressed in both ectopic and eutopic endometrium and stimulated inflammatory cells to secrete more interleukin-6 (IL-6), interleukin-8 (IL-8), COX-2 and other inflammatory mediators in EMs patients el. suggested that lipopolysaccharide (LPS) stimulated the production of high levels of TNF-α, IL-6 and IL-8 through activating the NF-κB pathway in the ectopenic endometrium stromal cells PC is the dry sclerotium of Polyporaceae Rosaceae plant peach or mountain peach, which was first recorded in Shen Nong's Herbal Classic. The main efficacy of PC includes promoting blood circulation, removing blood stasis, relaxing bowels as well as relieving cough and asthma  el confirmed that water extract derived from PK could effectively inhibit platelet aggregation and it was much more effective than the amygdalin and the fat oil of PK PK is the dried mature seed of Paeonia lactiflora is rhizome, which is divided into RPR and Radix Paeoniae Alba (RPA). TCM theory consider that RPA is beneficial to nourish yin and tonify deficiency, while RPR is suitable for promoting blood circulation, removing blood stasis and relieving pain Zheng Jia is blood stasis syndrome, the Paeonia lactiflora in GFPs should refer to RPR. Pharmacological researches indicated that the main components of RPA and RPR were monoterpene, glycoside, tannin and organic acids The medicinal part of Shen Nong's Herbal Classic. Its medical effectiveness is clearing heat and cooling blood, promoting blood circulation and removing blood stasis CM was first published in In the clinical treatment of EMs, GFPs is often changed into decoction, and the proportions of CT, PC, PK, RPR, CM is equal and the usual dose of five Chinese herbs in the prescription is 12 g el.In recent years, a series of studies proved that treating EMs with GFPs alone or in combination with western medicine had achieved satisfactory curative effect and was considered as an alternative drug for treating EMs AM is a common gynecologic disease, which is caused by invasion of endometrial glands and stroma into myometrium to form diffuse or localized lesions TCM has the characteristics of multi-component, multi-target and multi-pathway, as a result, it is difficult to analyze the complex mechanism of TCM only by traditional experimental methods http://tcmspw.com/tcmsp.php) and SymMap (http://www.symmap.org/). Second, 304 endometriosis-related genes were obtained from databases DisGeNET (http://www.disgenet.org/) and GeneCards (https://www.genecards.org/). Subsequently, 26 intersection genes were obtained by Venn diagram. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was explored to speculate the potential pathway of GFPs treatment for EM. Table Based on the principle of network pharmacology, we explored the mechanism of GFPs therapy for EMs. Firstly, we confirmed 102 targets of GFPSs from websites TCMSP (Treatment of EMs is a challenge in the field of gynecology. The accompanying dysmenorrhea and infertility have troubled women of childbearing age. However, the current treatment approaches have the defects of side-effect and high recurrence rate. Therefore, it is essential to develop drugs that can target the pathogenesis of EMs and effectively relieves the symptoms of patients without affecting ovulation and pregnancy functions. TCM is a precious resource, and many Chinese herbal medicines are worth exploring. Many clinical practices have proved the advantages of TCM in the treatment of disease, and have gradually attracted worldwide attention. EMs syndrome differentiation belongs to the category of blood stasis syndrome in TCM. GFPs has been used for the treatment of EMs in recent years as a famous classical Chinese medicine prescription commonly applied for the treatment of women's blood stasis disease in history. In order to deeply understand the effective components, mechanism and advantages of GFPs in clinical treatment of EMs, the articles concerned with pharmacology, animal experiments and clinical trials are classified and sorted out. At present, the main functions of medicine monomer in GFPs were focused on anti-tumor, anti-inflammatory, analgesic and hemodynamic improvement. The network pharmacology research also indicated that the potential mechanism of GFPs in the treatment of EMs might be inflammatory pathways, such as, TNF signaling pathway and NF-kappa B signaling pathway. However, it did not fully clarify the mechanism of GFPs as EMs therapeutic drug. For example, the understanding of medicine composition is insufficient. The related researches on medicine ingredients are few and lack pertinence to EMs. The clinical control experiment cases are few. Currently, more and more attention is being paid to the multidisciplinary integration research of TCM. For example, on the basis of classic famous prescriptions of TCM and combined with modern pharmacological research and evidence-based medicine, innovative addition and subtraction of Chinese herbs were carried out. The clinical efficacy of Sanjie Zhentong Capsule which is established by clinical experiences had been proved to be effective in the treatment of EMs


The vertebral column, or spine, provides mechanical support and determines body axis posture and motion. The most common malformation altering spine morphology and function is adolescent idiopathic scoliosis (AIS), a three-dimensional spinal deformity that affects approximately 4% of the population worldwide. Due to AIS genetic heterogenicity and the lack of suitable animal models for its study, the etiology of this condition remains unclear, thus limiting treatment options. We here review current advances in zebrafish phenogenetics concerning AIS-like models and highlight the recently discovered biological processes leading to spine malformations. First, we focus on gene functions and phenotypes controlling critical aspects of postembryonic aspects that prime in spine architecture development and straightening. Second, we summarize how primary cilia assembly and biomechanical stimulus transduction, cerebrospinal fluid components and flow driven by motile cilia have been implicated in the pathogenesis of AIS-like phenotypes. Third, we highlight the inflammatory responses associated with scoliosis. We finally discuss recent innovations and methodologies for morphometrically characterize and analyze the zebrafish spine. Ongoing phenotyping projects are expected to identify novel and unprecedented postembryonic gene functions controlling spine morphology and mutant models of AIS. Importantly, imaging and gene editing technologies are allowing deep phenotyping studies in the zebrafish, opening new experimental paradigms in the morphometric and three-dimensional assessment of spinal malformations. In the future, fully elucidating the phenogenetic underpinnings of AIS etiology in zebrafish and humans will undoubtedly lead to innovative pharmacological treatments against spinal deformities. The vertebral column (VC) or spine is the main feature that defines vertebrate animals. It extends from the head to the pelvis and consists in a straight segmented structure formed by a series of stacked bones or building blocks called vertebrae. These bones are separated and supported by intervertebral disks, muscles and ligaments. Morphologically, each vertebra is composed of a vertebral body or centrum, which wraps the notochord, and vertebral arches . This stAlong the antero-posterior axis, the human spine is divided into four portions or curves: cervical, thoracic, lumbar, and sacral. The thoracic and sacral portions are formed during the early prenatal period as a continuous curve, while the cervical and lumbar curves appear in the fetal stage. Thus, completing their development after birth in response to head movements and to sitting and walking positions . AbnormaScoliosis is the most common deformity of the VC in humans and represents a major public health issue . In humaWhile developmental and morphological differences exist between spines from distinct vertebrate species, a common evolutionary origin has been proposed for this skeletal structure. Indeed, the straight morphology of the VC is conferred by conserved molecules and signaling pathways orchestrating the establishment and maintenance of the spine 3D axial plane . HoweverDanio rerio) phenotyping data is providing a comprehensive collection of tools and information to understand spinal deformities in vertebrates, such as AIS. Mutant phenotypes displaying body axis curves are valuable genetic resources to identify molecular and cellular mechanisms that shape spine formation and patterning. Human genomic resources, coupled with phenogenetic information generated by gene function studies in zebrafish, will reveal novel genotype-phenotype correlations during spinal architecture morphogenesis and maintenance.In this review, we will discuss how the current emergence of zebrafish  was described as a familial condition, because of its high incidence among relatives . StudiesAlthough the idea that genetic factors contribute to scoliosis has strong supporting evidence, AIS inheritance does not behave as a simple Mendelian mechanism. It is thought that AIS is a polygenic condition with genetic heterogenicity, variable patterns of inheritance, and a mode of genetic transmission that still remains to be identified . Even coTo study the causes of AIS in humans, diverse animal model systems have been used to date. Due to the low natural incidence of scoliosis in quadrupedal organisms, it has been proposed that upright position is critical in the development of spinal malformation . Also, tBecause of the lack of non-human bipedal mammals and the low rate of spinal deformities in quadrupedal vertebrates, researchers have developed several procedures to induce bipedalism and scoliosis-like phenotypes in experimental animals. These include amputation of forelimbs and tail, or pinealectomy  axis from the paraxial mesoderm, which undergoes mesenchymal-to-epithelial transition, and is patterned by maternally defined signaling morphogen gradients such as Nodal, Wnt, and FGF . Somitesectonucleoside triphosphate diphosphohydrolase 5 (entpd5) gene in the notochord sheath cells, and relies on the Notch pathway. Entpd5 functions to recruit osteoblasts to the mineralized zone and ultimately form the centra  that surround the notochord sheath . The choe centra . Dependie centra .Although the spine is formed in all vertebrates from the sclerotome and notochord, there are important differences concerning the contribution of each embryonic tissue and the concomitantly spine formation between species. Some of these include the proportion of the somites, the segmentation pattern of the spine that relies on the segmentation clock patterning, and the segmental periodicity of vertebrae that is determined by the sclerotome instead of the notochord in some species. However, in teleost fish, the segmentation of the spine relies on the notochord, with contribution from the paraxial mesoderm . DespitePhenotyping efforts in zebrafish based on forward and reverse genetic approaches , along wIn the zebrafish early embryo, notochord formation is critical for the AP extension of the body axis, and ultimately, spine biogenesis. Signaling pathways, such as Notch, regulate notochord-lineage cell fate by determining if these differentiate into chondrocytes or chondroblasts . As aforThe study of a collection of zebrafish mutants displaying scoliosis-like phenotypes has significantly increased our knowledge regarding the genetic regulation of spine development . A forwaleviathan, which shows a dorsoventral and lateral folded notochord  gene, which functions in the cilia assembly, was identified in skolios mutants (kif6 mutants (protein tyrosine kinase 7 (ptk7) gene indicates a role of the Wnt/β-catenin and planar cell polarity signaling in patterning, establishing and maintaining axial coordinates during the juvenile-to-adult transition  gene leads to failure in notochord function, which generates a scoliosis-like phenotype , indicates a role of Smt/Dstyk in establishing spine axial coordinates through a lysosome-to-nucleus signaling mechanism -based mutagenesis screens and more recently, by specifically mutating cilia-related genes . Thus, mkurly/cfap298 mutant), allowing the embryo to develop normally when the temperature is shifted from a warmer to cooler conditions. These animals appear wild-type through the first development stages but develop spinal curves during late larval stages. The second strategy consisted of injecting wild-type RNA at 1-cell stage in animals from three different mutant backgrounds showing defective cilia motility. This rescue approach allowed early development to proceed normally, and revealed that older embryos developed curved spines  promotes primary cilia assembly and localizes at centrioles and basal bodies. Mutations in this gene have been implicated in different human ciliopathies, including Joubert syndrome, which is characterized by hindbrain malformation, and motor and cognitive impairments . Zebrafid vision . Additioto breed , thus suarmc9 crispant shows a decreased number of cilia in brain ventricles, and typical ciliopathy phenotypes, including scoliosis-like spinal curvature  protein has recently been associated with primary cilia function and ciliopathies . Armc9 lurvature .The role of cilia in spine coordinate establishment and morphology is increasing the spectrum of genotype-phenotype relationships in vertebrate post-embryonic development so far. The use of zebrafish seems advantageous in the study of primary and motile cilia structure and how their functions contribute to body axis development and the maintenance of its straightening. Ongoing phenotyping projects in zebrafish are expected to provide unprecedented genetic entry points to cilia biology and spine morphogenesis.CSF is a clear liquid filling the cerebral ventricles and central canal of the spinal cord. This fluid also surrounds the central nervous system (CNS) in the subarachnoid space . CSF funThe “classic CSF circulation theory” presumes a constant downward flow from the lateral ventricles toward the subarachnoid space. This theory describes CSF flow as unidirectional, circulating from the lateral to the third ventricle via the foramen of Monro, and then through the Sylvian aqueduct to the fourth ventricle. Subsequently, the CSF flows caudally along the central canal of the spinal cord or circulates through the foramens of Luschka and Magendie into the subarachnoid space of the brain and spinal cord, where a portion of CSF drains to the blood via arachnoid granulation, via spinal nerve roots and the remainder via the olfactory tracts .Recently, the use of molecular, cell biology, and neuroimaging techniques has revealed that CSF flow is not as simple as previously thought. This flow depends on multiple factors including cilia motility, hearth beat, and pulsatile and local exchange between interstitial fluid, blood, and CSF . AlthougIn vertebrates, the central canal of the spinal cord is a narrow cylindrical cavity surrounded by ependymal cells. It extends caudally from the floor of the fourth ventricle to the terminal ventricle or ampulla caudalis, at the conus medularis . The cenIn addition to the CSF, the central canal contains a threadlike structure, called Reissner fiber (RF) . This fiIn zebrafish, CSF circulation in the spinal cord is critical for spine morphogenesis, and disturbances in its dynamics have been associated with scoliosis . The relptk7 mutant shows abnormal cilia structure and organization, as well as CSF flow defects, hydrocephalus and scoliosis   , and actorphants . These forphants .Secondly, CSF flow is necessary for the correct aggregation of RF components. RF is an elastic extracellular structure suspended in the CSF and extended from the diencephalon to the entire length of the central canal of the spinal cord  Rodrigu. RF is escospondin (sspo) mutant that cannot assemble the RF. Such a mutant embryo develops a curly tail down phenotype, which gradually worsens until death occurs 10 days after fertilization , at the same time that appears the curved axis phenotype  along the entire nervous system . DependiThe transparency of the zebrafish larvae facilitates the visualization of CSF flow, RF dynamics and CSF-cNs connectivity, elements that conform a proprioceptive organ involved in locomotion and postural control and is also required for the maintenance of natural curvature of the spine . The preOn the other hand, there are arguments suggesting that in humans this proprioceptive organ would not be present, or it will have some differences. Firstly, the central canal of human spinal cord, in embryos and children, is similar to the one described in other vertebrates . After tIn spite of these differences between humans and other vertebrate organisms, there are also antecedents suggesting that cilia and spinal CSF flow are important to maintain the spinal curvature and that disorders in the proprioception are a cause of AIS. A recent study using a noninvasive and radiation-free MRI revealed disturbances in the CSF flow into the spinal cord in scoliotic individuals, especially at the thoracic level . This fiA compromised CSF flow in human scoliosis has also been suggested in individuals with Chiari malformation type I (CM-I) with or without syringomyelia . Indeed,In relation with the link between cilia and scoliosis in humans, is important to separate between motile cilia and primary cilia disturbances. In this way, PCD is characterized by an alteration in motile cilia, leading to chronic respiratory tract infections, abnormally positioned internal organs, and infertility   gene . This getructure . By cont(TTLL11) . This pr(TTLL11) .In summary, sequence alterations of these three genes  found by WES analysis from scoliotic individuals suggest disturbances of primary cilia function. The posterior generation of zebrafish mutants confirmed the relevance of these genes in the maintenance of a straight body axis. In this way, the massification of WES analysis and the ease performing of CRISPR/Cas9-mediated targeted mutagenesis in zebrafish, are providing us with a powerful tool to understand the pathophysiology of human diseases, particularly scoliosis.Other mutations affecting primary cilia function , the incidence of scoliosis and the severity of spinal curvature were considerably reduced  treatment, suggesting that an oxidative stress-dependent immune response controls the degree of scoliosis in zebrafish  and post-embryonic stages  inhibitor can fully rescue normal axial development, even though the same animals developed scoliosis during early juvenile stages  will provide new ways of preventing and treatment for AIS in humans.Currently, the availability of complete genome sequences of many model organisms is allowing us to evaluate gene function and decipher the complex crosstalk between genomics and phenomics. In this quest, deep phenotyping is a valuable approach to capture subtle and dynamic cellular and molecular variations . This coTraditional and automated histological techniques such as high-throughput histology and micron-scale computed tomography (microCT) have recently been used to describe mutant phenotypes, including those showing alterations in skeleton morphogenesis. Thus, whole-mount staining and high-resolution imaging of juvenile and adult animals are being exploited to generate precise phenogenetic information of organ systems and tissue morphology as well as physiological defects at cell resolution level, including the spine architecture . This, hAlthough microCT imaging has allowed quantitative morphometric analysis of the spine defects in zebrafish AIS-like models, it has also revealed subtle phenotypic deviations that would not be observable by the naked eye. The study of post-embryonic features by using deep skeletal phenotyping identified spatial bone mineralization patterns within the axial skeleton . For insIn addition to mutagenesis approaches, complementary high-resolution and -quality 3D axial skeleton reconstructions will help determine the morphological effects of manifested mutations generating spine malformations . ImportaScoliosis is the most common deformity of the spine in humans. Among the different types of scoliosis, AIS represents a predominant spine deformity worldwide. Since the first report of AIS in humans , our knoSeveral mechanisms and features of zebrafish spine deformity, such as the role of ciliogenesis, CSF flow, and inflammation, have been investigated in mutants to particular human AIS-related genes. Although the contribution of zebrafish as a genetic system has been crucial to study spine malformation, it is unclear if this animal model recapitulates the pathogenesis cause for all known human AIS risk loci. Further knowledge of the genetic factors controlling the spine formation is critical to understand the etiology of AIS. By doing so, it will reveal candidate genes associated to this malformation and allow the development of new therapeutic alternatives to improve the quality of life of humans worldwide.The progress in the study and definition of genetic risk factors for human scoliosis appears to be limited in the ability to link specific genomic sequences with the disease, which could increase clinical biases and imprecise genotype-phenotype associations. High throughput next-generation sequencing has unveiled the dysregulation of thousands of non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), linked to scoliosis . Indeed,In addition, the integration of disciplines, such as genomics and phenomics, into the study of zebrafish spine malformations will provide a description of the molecular, morphological, physiological and behavioral underpinning scoliosis/AIS. This will allow us to construct molecular genotype-phenotype maps that would be valuable resources to investigate not only this condition, but also other human diseases. In light of this, further efforts to construct molecular genotype-phenotype maps in zebrafish will provide guiding principles into human scoliosis/AIS. Taken together, we foresee that by using zebrafish, along with phenogenetic approaches would then in turn allow to progressively conduct novel therapies to treat scoliosis in humans.