Furthermore, it has been demonstrated that autophagy and exosomes may be concomitantly or reciprocally regulated, depending on the cellular conditions. Given that increased levels of autophagy are required to activate HSCs, it is important to investigate whether autophagy levels decrease at later stages of hepatic stellate cell activation, leading to increased release of exosomes and further propagation of hepatic fibrosis.Chronic liver injury, regardless of the underlying disease, results in gradual alteration of the physiological hepatic architecture and in excessive production of extracellular matrix, eventually leading to cirrhosis Liver cellular architecture consists of different cell populations, among which hepatic stellate cells (HSCs) have been found to play a major role in the fibrotic process. Under normal conditions, HSCs serve as the main storage site for vitamin A, however, pathological stimuli lead to their transdifferentiation into myofibroblast cells, with autophagy being the key regulator of their activation, through lipophagy of their lipid droplets. Nevertheless, the role of autophagy in liver fibrosis is multifaceted, as increased autophagic levels have been associated with alleviation of the fibrotic process. In addition, it has been found that HSCs receive paracrine stimuli from neighboring cells, such as injured hepatocytes, Kupffer cells, sinusoidal endothelial cells, which promote liver fibrosis. These stimuli have been found to be transmitted  Liver fibrosis is a homeostasis disorder defined by increased synthesis and deposition of extracellular matrix (ECM) and a parallel decrease of physiological mechanisms underlying matrix degradation or remodeling . Liver iFibrosis progression results from excessive ECM deposition, predominantly produced by activated hepatic stellate cells (aHSCs), and failure of matrix degradation . In the Liver fibrosis is a prolonged wound healing process characterized by distorted hepatic architecture and excessive ECM accumulation, produced by a heterogeneous population of myofibroblasts, such as HSCs, bone-marrow derived fibrocytes, portal myofibroblasts and epithelial to mesenchymal transition . Myofibrvia contraction or dilation of the sinusoidal lumen  and have a starlike configuration due to their dendritic cytoplasmic extensions, which let them interact with hepatocytes, adjacent HSCs and LSECs  and are al lumen . Moreoveal lumen . These sal lumen . The rolAs a result of disturbances in hepatic homeostasis after liver injury, qHSCs undergo “activation” or transdifferentiation, from a quiescent, vitamin A –storing cell to a myofibroblast-like cell, with the loss of lipid droplets being a hallmark of HSCs activation. During activation, qHSCs acquire several new phenotypic characteristics, such as expression of α-Smooth muscle actin (αSMA), fibrillary collagens (mainly type I and type III), increased expression of tissue inhibitors of metalloproteinases (TIMPs), platelet-derived growth factor receptor beta (PDFGR-β), transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) . ConsequActivation of HSCs consists of two phases; initiation and perpetuation . InitiatIn vitro cultures of HSCs have demonstrated morphological differences between initiation and perpetuation HSCs, including the evidently higher levels of a-SMA protein expressed in perpetuation HSCs. Moreover, the same study has shown that molecules derived by initiation HSCs seem to protect hepatocytes form cell death, while on the other hand perpetuation HSCs exhibited active synthesis and secretion of fibrogenic molecules  from damaged neighboring cells, such as apoptotic hepatocytes, KCs, LSECs and platelets, as well as changes in the surrounding extracellular matrix, result in genetic and transcriptional modifications, rendering HSCs more responsive to cytokines and stimuli. Induction of the expression of PDGFR-β in aHSCs is a hallmark of early activation and is followed by development of contractile and fibrogenic phenotype . Moreoveolecules , secrete process .Resolution of the injury stimuli may result in attenuation or even regression of liver fibrosis by inducing cell death, senescence or regression of aHSCs to their quiescent phenotype .Autophagy is an evolutionarily conserved catabolic process in which intracellular macromolecules and organelles (referred to as “autophagic cargo”) undergo lysosomal degradation. There are 3 main types of autophagy: macroautophagy (thereby referred to as “autophagy”), microautophagy and chaperone-mediated autophagy, all of which differ in their delivery methods to the lysosome . AutophaMeasuring the autophagic flux is of great interest since autophagic levels affect the function and the structure of the cell and the whole tissue. The cytosolic form of MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3), termed LC3-I, is lipidated to form LC3–phosphatidylethanolamine, LC3-II, which is specifically recruited to the phagophore membrane. LC3-II, present in the inner membrane of the autophagosome remains conjugated during the autophagosome maturation, thus levels of LC3-II correlate well with the autophagic flux . Degradavia direct phosphorylation and inactivation of Tuberous sclerosis complex (TSC) (inhibitor of mTORC1).The main molecular signaling pathway by which autophagy exerts its multifunctional role during liver fibrosis is through Phosphoinositide 3-kinase/Protein kinase B (PKB), also known as Akt/mammalian Target of Rapamycin (PI3K/Akt/mTOR)  inhibits autophagy . The impin vivo and in vitro to further investigate the role of autophagy in HSCs activation and transformation into a myofibroblast like cell  and long-non-coding RNAs (lncRNAs) and they can be transferred from donor cells to recipient cells, where they can activate or regulate cell activities such as protein expression, cell proliferation and differentiation or metabolic alterations . ExosomeExosomes are spherical to cup-shaped membranous vesicles with a size of 30–100 nm. They are formed by inward protrusions of the limiting membrane of early endosomes as small intraluminal vesicles (ILVs), which mature into late endosomes/multivesicular bodies (MVBs) . MVBs maExosome biogenesis is associated with two distinct trafficking pathways; ESCRT - dependent and ESCRT independent pathways . There aESCRT independent pathways involve exosome biogenesis in the absence of the ESCRT protein machinery . CeramidThe fate of MVBs is either the degradation pathway, mediated by fusing with the lysosome compartment or the secretion pathway, with MVBs being released as exosomes. Exosome release appears to be controlled by RAB guanosine triphosphates GTPases. RAB5 is associated with early endosomes, while RAB7 is involved in the formation of late endosomes, by regulating the fusion of MVBs with lysosomes. On the contrary RAB27 regulates the docking and fusion of MVBs with the plasma membrane . On the Following their release into the intercellular space, exosomes are internalized by target cells by three mechanisms: docking to membrane receptors, fusing directly with the plasma membrane, or following the endocytic pathway  thereby Potential regulation of exosome secretion by mTOR signaling pathway has emerged as a novel research field in liver fibrosis, however, whether it inhibits or induces secretion remains controversial. Exosomes are considered both as an intercellular communication mediator and as an alternative pathway to eliminate excessive and damaged cellular components. It has been demonstrated that exosome release is regulated by mTOR pathway in response to changes in nutrient and growth factors, as well to stress exposure . Under avia exosomes  from MVBs, thus preventing the acidification of MVB lumen and allowing fusion with the plasma membrane. Hence the luminal pH of the MVBs plays a key role in controlling whether MVBs undergo lysosomal degradation or plasma membrane fusion. Moreover LC3-II has been demonstrated to be located on the lumen side of MVBs, while intact LC3-II positive exosomes are released in the extracellular environment (Emerging evidence suggests direct links between autophagy and exosome biogenesis through intertwined molecular machinery. The autophagy-related proteins (ATG) 5 and 12 (ATG5-ATG12) and the autophagy-related 16 like 1 (ATG16L1) complex, along with LC3-II, have been observed to localize to endosomes and have a curtail role in exosome biogenesis . On thisironment . Anotherironment .Autophagy levels affect the number of secreted exosomes, as it has been demonstrated that autophagy and exosomes are reciprocally regulated. Inhibition of autophagy lead to increased exosomal secretion, while autophagy induction prevented extracellular release of exosomes .via the endocytic pathway could follow two possible pathways: degradative or secretory (via exosomes, which escape from the degradative lysosomal pathway (Exosomes which are internalized by target cells ecretory . Exosomeecretory . Amphisoecretory . On the ecretory . On the  pathway .The vesicular interaction between autophagy and exosomes has also been demonstrated in the liver. It has been shown that during liver fibrosis exosomes released from injured LSECs and Kupffer cells contain high levels of PDGF molecules . In turnIntriguingly emerging evidence indicates that exosomes could also affect autophagic levels by encapsulating autophagic proteins as their cargo, such as SQSTM1, LC3  or autopThe effect of exosomes on autophagy depends on its parental cells and the conditions that stimulate their production. More specifically, evidence has been provided that exosomes secreted by mesenchymal stem cells can induce moderate autophagy on target cells, thus playing a protective role, while on the other hand exosomes produced by injured cells can transfer harmful substances, induce inappropriate autophagy, and damage normal cells . In factLiver constitutes a complex and multicellular organ; thus, maintenance of homeostasis is crucial to ensure proper function. Autophagy plays a key role in the homeostatic process through the degradation of protein aggregates and dysfunctional organelles . MoreoveLiver fibrosis, regardless of the etiologic factor, is characterized by cell death of hepatocytes and suppression of liver regeneration, resulting in replacement of normal liver tissue by accumulated ECM . Concomiin vitro in human tissue and ex vivo in rodent models of liver injury, by a significant increase of autophagic vacuoles, LC3-II levels and autophagic flux (Activation of HSCs consists of two distinct phases; initiation and perpetuation . During gic flux . Howevergic flux .On the other hand, once HSCs are activated and become transdifferentiated into myofibroblast-like cells, they probably no longer confront stressful conditions, neither increased energy demands as they have been adapted to their new “normal” situation, and their current activity is to promote ECM accumulation and liver fibrosis . The dowThe pivotal role of autophagy and exosomes and their multifaceted crossregulation during the different stages of activation process remain a controversial research topic. Up to date, studies have shown that induced autophagy of aHSCs in fibrotic liver seems to act as an inhibitor of the fibrotic process by promoting cell death of aHSCs . HoweverAC conceived and designed the review, studied the literature, wrote the manuscript, and revised the schemes. EM studied the literature, wrote the manuscript, and drew the schemes. AG, GG, and PK studied the literature, revised the manuscript critically for its content and performed modifications. All authors accepted the final version of the review and approved it for publication.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.


Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating  Tooth development in mammals is guided by reciprocal and dynamic interactions between the oral ectoderm and the underlying neural crest-derived mesenchyme. These interactions are mediated by many secreted signaling molecules and transcription factors, which are expressed in specific patterns. Signaling molecules involved in the process include fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), wingless-type MMTV integration site family (WNT), and sonic hedgehog (SHH). The transcription factors involved include members of msh homeobox (MSX), distal-less homeobox (DLX), paired box (PAX), and LIM homeobox proteins (LHX) ,2,3.The first morphological sign of tooth development appears as localized thickening of the oral ectoderm  at the 11th day of mouse gestation (E11). The epithelium further thickens and invaginates into the mesenchyme to form a dental placode E12) and then a bud E13), with the mesenchyme cells condensing around the bud. Subsequently, a signaling center called an enamel knot is established in the epithelium, and it directs further morphogenesis into the cap stage (E14.5) and the bell stage (E16.5). Differentiation of ameloblasts and odontoblasts from the epithelium and the mesenchyme, respectively, occurs during the late bell stage (E18.5) . We. WeRspo3Rspo2 and Rspo3 during craniofacial development in mammals. A recent publication showed that Rspo2 and Rspo3 synergistically regulated craniofacial and dental development in fish [Rspo genes, which made it important to examine mouse mutants. While further investigation is necessary to determine the molecular and cellular changes in the mouse double mutants, our data establish that Rspo3, along with Rspo2, is a crucial regulator of oro-facial development from early stages.The current study also provides the first description of the phenotypic consequences from the simultaneous loss of  in fish . HoweverRspo3 is important for development of the lower incisors in mammals, regulating the bud-to-cap transition. Furthermore, Rspo2 and Rspo3 have overlapping yet crucial roles in early development of the face in mammals.


Coating magnesium alloys with nitride surface layers is a prospective way of improving their intrinsically poor surface properties; in particular, their tribological and corrosion resistance. These layers are usually produced using PVD methods using magnetron sputtering or arc evaporation. Even though the thus-produced layers significantly increase the wear resistance of the alloys, their effects on corrosion resistance are unsatisfactory because of the poor tightness, characteristic of PVD-produced products. Tightness acquires crucial significance when the substrate is a highly-active magnesium alloy, hence our idea to tighten the layers by subjecting them to a post-deposition chemical-hydrothermal-type treatment. This paper presents the results of our experiments with a new hybrid surface engineering method, using a final tightening pressure hydrothermal gas treatment in overheated steam of the composite titanium nitride layers PVD, produced on AZ91D magnesium alloy. The proposed method resulted in an outstanding improvement of the performance properties, in particular resistance to corrosion and wear, yielding values that exceed those exhibited by commercially anodized alloys and austenitic stainless 316L steel. The developed hybrid method produces new, high-performance corrosion and wear resistant, lightweight magnesium base materials, suitable for heavy duty applications.  This creates the need for new, effective surface engineering solutions that would increase the application potential of such alloys. In addition to the high specific strength, the attractiveness of magnesium alloys lies in the unique combination of their low density and a range functional properties, such as effective screening of electromagnetic fields, good vibration damping, biocompatibility, and excellent castability, which allows mass production of complex details in a single die casting operation. The barrier for wide expansion of magnesium alloys is their poor performance properties, in particular low corrosion and wear resistance. Unlike aluminum or titanium alloys, no magnesium alloys that are resistant to corrosion in chloride environments are available. The increasing tendency to reduce the weight of products by making wider use of lightweight materials, driven by practical, economic, and environmental considerations  poses new challenges by putting new demands on magnesium alloys, which stimulate their dynamic development : 2 × 10 × 3). Before the deposition of each subsequent coating, oxide passivation film was removed from the surfaces via ion etching in cathode sputtering process in the processing chamber. The intermediate layers, counting from the substrate: aluminum and titanium produced in laboratory magnetron sputtering device, working in DC mode, using aluminum and titanium targets of 99.5 and 99.6 purity, respectively (GfE GmbH), and the external layer of TiN-type titanium nitride layer in the arc evaporation MZ383 device  manufacturerin a reactive process using titanium targets of 99.6 (GfE GmbH) and nitrogen atmosphere with 6N purity. The detailed parameters used in the each of the PVD process stages, developed in previous works , are givThe final stage of the hybrid method consisted of sealing the layers by pressure hydrothermal gas treatment performed in overheated steam . The treIn order to assess how well the examined pressure hydrothermal gas treatment variant of the composite titanium nitride layers improved the performance properties of the AZ91D alloy (TiN2Ti1Al10_SS), several other coating–substrate variants and other reference materials, listed with their denotations in +) primary beam and secondary ions as measured, as MeCs+ clusters were analyzed. The method of measuring oxygen as OCs+ clusters has been described elsewhere [The characterization of the composite TiN-Ti-Al type titanium nitride layers’ microstructure involved cross section optical microscopy examinations, scanning electron microscopy (SEM) observations of the layers surface morphology  and secondary ion mass spectrometry (SIMS) chemical composition analyses . SIMS measurement was conducted using a cesium (Cslsewhere . The chelsewhere . The eff3) and a heat-treated 1045 steel disk  as the counter body. The tests were performed within a load range up to 200 N with a rotational speed of 200 revolutions/min for 1 h.The corrosion resistance of the composite titanium TiN-Ti-Al surface layers that were processed hydrothermally in overheated steam, as well as those of other reference materials , were exThe microstructure of the composite surface titanium nitride TiN–Ti–Al type layers with a sub-layer of titanium and aluminum (TiN2Ti1Al10), produced on AZ91D magnesium alloy using the hybrid method, combining magnetron sputtering of intermediate layers and cathode arc evaporation of the outside titanium nitride layer, is shown in The thickness of the component layers starting from the surface was successively: surface titanium nitride layer (gold)—circa 2 µm; titanium sub-layer (grey)—circa 1 µm and aluminum (white)—circa 7 µm . Some deThe hydrothermal treatment in overheated steam, as shown in Ra (average arithmetic deviation of the roughness profile from the median line) 160 nm for the as-deposited layer variant (TiN2Ti1Al10), up to Ra 185 nm for the overheated steam variant (TiN2Ti1Al10_SS) and Ra 210 nm for the boiling water bath variant (TiN2Ti1Al10_S). This is due to the oxide film that formed during the hydrothermal treatment. However, although the oxide film produced in overheated steam slightly increased the surface roughness of the layers, its roughness was still lower (better for corrosion resistance) than that of the boiling water variant.Profilometry measurements indicated that the surface roughness changes from The distribution of the elements in the TiN-Ti near-surface zone of the composite TiN2Ti1Al10-type titanium nitride layers, determined using secondary ion mass spectrometry (SIMS), is shown in Ecor,icor) values, as well as the breakdown potential (Enp) obtained by potentiodynamic examination, are shown in Ecor = −900 mV), and so was expected to improve the corrosion resistance as a sub-layer in the composite TiN-Ti-Al titanium nitride layer structure, acting as an anti-corrosive barrier able to stop the access of the aggressive environment to magnesium alloy. It was only after the final hydrothermal treatment in boiling water bath was performed [Ecor = 1300 mV  with Ecor = −1530 mV. The resulting increase of corrosion potential is circa ΔEcor = 220 mV higher than in the case of hydrothermal treatment in boiling water bath and is associated with the corrosion current decrease by an order of magnitude at circa icor = 0.022 µA/cm2. This demonstrates a more efficient tightening of the layer in the overheated steam process. The main benefits from processing in overheated steam is the resulting relatively broad (ca. 300 mV large) passive range with a positive breakdown potential of circa Enp = 350 mV, resulting in corrosion resistance that is much better than that of the commercially anodized AZ91D alloy (ANOD)  shift in the corrosion potential to the value of circa y (ANOD) , which dss steel . The polss steel , showingThe outstanding improvement of corrosion behavior of the composite TiN-Ti-Al titanium nitride layers  achieved by using hydrothermal treatments in overheated steam (TiN2Ti1Al10_SS) described in the preceding section  and may Ra increased by circa 30% for the hydrothermal treatment in boiling water bath (TiN2Ti1Al10_S), compared to only circa 15% for the treatment in overheated steam (TiN2Ti1Al10_SS). The oxide layer thickness seems thus to be more uniform in the latter case. The lower surface development is also more favorable in terms of corrosion resistance. However, the most efficient improvement of corrosion resistance of the composite titanium nitride layer on AZ91D was observed in the case of hydrothermal processing in overheated steam and is certainly primarily related to the more effective tightening of the layer with titanium oxides produced in reaction with the overheated steam; not only on its surface, but most likely also inside the discontinuities related to droplets and craters penetrating into the depths of the layer. This is clearly confirmed by SIMS analysis  facilitates steam penetration deep into the composite titanium nitride layer along the discontinuities and resulting in oxidation; hence, titanium oxide also forms inside the layer and seals the discontinuities. The appearance of a passive range on the polarization curve in positive corrosive potential areas produced by treatment in overheated steam  is most In the case of the composite titanium nitride layer processed in the boiling water bath (TiN2Ti1Al10_S), as shown by SIMS examination , the tigThis leads to the conclusion that the effective sealing deep inside of the outside titanium nitride layer, and in particular of the titanium sub-layer, such as was obtained in the overheated steam hydrothermal treatment variant, is essential to effectively isolate the aluminum sub-layer on magnesium alloy from the environment, and therefore to eliminate the risk of galvanic cells formation to prevent the accelerated galvanic corrosion of the AZ91D alloy covered with the composite titanium nitride layer. Even though, contrary to the original assumptions , the aluA complex characterization of the mechanical performance properties of the composite TiN-Ti-Al type titanium nitride layers was performed during our previous work  and demoHigh mechanical resistance of the TiN-Ti-Al type layers guarantees high functional durability in those service conditions where composite titanium nitride layers on AZ91D magnesium alloy are exposed to both corrosive and mechanical factors. This is of crucial importance, taking the risk of galvanic corrosion in case of mechanical failure of the layers into account. What needs to be emphasized is the superiority of the variant of hydrothermal processing of the composite titanium nitride layers in overheated steam (TiN2Ti1Al10_SS) over treatment in a boiling water bath (TiN2Ti1Al10_S) in terms of mechanical durability of the layers. This is related to the fact that in this case the corrosion resistance, as discussed above, is controlled by the tightness of the titanium sub-layer, which is less sensitive to any damage that could occur in the titanium oxide film tightening the surface or, even more, in the titanium nitride outer zone.Corrosion resistance of the composite TiN-Ti-Al titanium nitride layers with titanium and aluminum sub-layers produced on AZ91D magnesium alloy using the PVD hybrid method, may be radically improved by applying a final tightening procedure using a pressure hydrothermal gas treatment performed in overheated steam (recently patented), which results in a better corrosion resistance than that of 316L stainless steel.The outstanding improvement of the composite TiN-Ti-Al titanium nitride layers corrosion resistance processed in overheated steam may be attributed to the effective deep sealing inside the layers, and in particular, on the level of the titanium sub-layer, which actually became a corrosion barrier controlling the behavior of the entire layer. Moreover, as a direct consequence, the as-sealed composite layer has the crucial advantage of reducing the risk of galvanic corrosion once any mechanical damage in service conditions occurs in the titanium oxide film tightening the surface of the layer or in the titanium nitride outer zone, breaking their continuity. The susceptibility to galvanic corrosion increases when the corrosion barrier is limited to a surface titanium oxide film, like the one produced using the originally developed hydrothermal treatment variant in a boiling water bath.The corrosion resistance of AZ91D alloy covered by the composite TiN-Ti-Al titanium nitride layers hydrothermally sealed in overheated steam is significantly higher than that of the commercially anodized AZ91D alloy, and also much higher than when composite TiN-Ti-Al titanium nitride layers are tightened by hydrothermal treatment in a boiling water bath. The key advantage of the composite titanium nitride layers on the AZ91D alloy that is hydrothermally sealed in an overheated steam is their passivity in the relatively large range with the positive breakdown potential, obtained thanks to the anticorrosion properties of the effectively sealed titanium sub-layer.In addition to its high corrosion resistance, the composite titanium nitride layers on the AZ91D alloy exhibit high resistance to wear which significantly exceeds the wear resistance of 316L stainless steel and anodized AZ91D alloy. The newly developed hybrid method that involves a key operation of final sealing of the nitride layers in overheated steam therefore allows to produce high performance, both corrosion and wear resistant, lightweight magnesium base material suitable for wider applications.


Multipotent mesenchymal stromal cells (MSCs) are promising cellular therapeutics for the treatment of inflammatory and degenerative disorders due to their anti-inflammatory, immunomodulatory and regenerative potentials. MSCs can be sourced from a variety of tissues within the body, but bone marrow is the most frequently used starting material for clinical use. The chemokine family contains many regulators of inflammation, cellular function and cellular migration–all critical factors in understanding the potential potency of a novel cellular therapeutic. In this review, we focus on expression of chemokine receptors and chemokine ligands by MSCs isolated from different tissues. We discuss the differential migratory, angiogenetic and immunomodulatory potential to understand the role that tissue source of MSC may play within a clinical context. Furthermore, this is strongly associated with leukocyte recruitment, immunomodulatory potential and T cell inhibition potential and we hypothesize that chemokine profiling can be used to predict the in vivo therapeutic potential of MSCs isolated from new sources and compare them to BM MSCs. Mesenchymal Stromal Cells (MSCs) are a non-hematopoietic multipotent adult progenitor population that were first named in 1976 by Alexander Friedenstein as colony-forming unit fibroblasts (CFU-Fs). These cells were spindle shaped, clonogenic in monolayer cultures and could serve as feeders in the bone marrow for hematopoietic stem cells . The terMSCs exert potent anti-inflammatory, immunoregulatory and pro-angiogenic effects through interactions with the immune system and the secretion of paracrine immunomodulators. These characteristics, coupled with their ease of accessibility and expansion resulted in a growing interest in the therapeutic potential of these cells. However, the variety of tissue sources, coupled with different isolation and culture protocols and the intrinsic variability of MSCs from donor to donor has led to wide variability regarding the description of MSC phenotypes and properties through the literature. To address this matter, the International Society of Cellular Therapy (ISCT) specified the criteria human cells had to reach in order to be defined as MSCs: plastic-adherence when maintained in standard culture conditions and specific surface antigen expression along with trilineage differentiation potential into osteoblasts, adipocytes and chondroblasts , 6. MSCsThese criteria have helped to drive standardization in the manufacturing and identity of MSC for therapeutic use, but does not capture whether MSCs derived and cultured from different tissues have equivalent therapeutic potential or potency. The chemokine family contains many regulators of inflammation, cellular function and cellular migration–all critical aspects in designing a novel cellular therapeutic and understanding potential potency. In this review, we focus on expression of chemokine receptors and chemokine ligands by MSCs isolated from different tissues.The use of MSCs as cellular therapeutics is limited by the low frequency of these cells within tissues and the high doses required for medical use. The American Code of Federal Regulation of the Food and Drug Administration and the European Medicines Agency have established guidelines, generally known as “Good Manufacturing Practice” (GMP), that cover cell culture procedures, reproducibility, efficiency, and safety. Most isolation and cell culture protocols are not optimal for GMP adaption, which has limited the tissue sources used for MSC isolation driving clinical use into narrow corridors . AccordiIt is therefore well established that MSCs can be isolated from most tissues within the body and that tissue source of origin as well as the conditions used to expand the cells influence MSC secretome, and thus, their therapeutic potential –13. For Due to their differentiation potential, the initial clinical use of MSC was aimed at tissue reconstruction or regeneration including musculoskeletal tissues, nervous system, liver and skin among others. However, this regeneration potential has only been clinically proven to be effective in MSC-based bone regeneration . Even inIn response to their microenvironment, MSCs modulate innate and adaptive immune responses  as well The mechanisms of migration of MSC to defined in vivo niches following systemic administration and persistence in these sites whether by homing or local administration remains largely unexplored. MSC migration and homing is hypothesized to be similar to leukocyte migration from the bloodstream and to involve adhesion molecules for rolling and trans-endothelial migration , 25; cheChemokines are a family of small heparin-binding homologous proteins involved in the regulation of cell migration under both inflammatory and physiological conditions . ChemokiChemokine nomenclature is based on their structural classification but can also be sorted out into functional categories; homeostatic, proinflammatory and multifunctional according to the microenvironment. Inflammatory chemokines become upregulated in the presence of inflammation, promoting the recruitment of leukocytes. As an example, all nucleated cells can upregulate the expression of CCL2 to induce the recruitment of leukocytes towards a site of injury or infection. Inflammatory chemokines and their receptors display complex interaction patterns; a single ligand has broad receptor selectivity and a single receptor has a broad number of agonists to enable a rapid immune response to protect the host . ChemokiThe expression of homeostatic chemokines is constitutive and cell or tissue specific . These cCell migration is induced upon interaction of chemokine ligands with their cognate receptors. Chemokine receptors are part of the γ subfamily rhodopsin-like seven-transmembrane receptors and, unlike their ligands, are well conserved among species. Chemokine receptors can be classified into G protein-coupled or classical chemokine receptors and atypical chemokine receptors (ACKRs) according to their dependency on G proteins for signaling and the atypical expression patterns . ClassicMSCs reside in specific tissue niches where their microenvironment regulates the balance of proliferation and differentiation, as well as their secretome, which includes cytokines, chemokines, immunomodulatory molecules, and growth factors . In thisInflammatory response to injury results in the secretion of an array of chemokines in a temporally and spatially orchestrated manner that results in cell migration. The secretion of CXCL8 soon after injury results in the recruitment of neutrophils to the damaged or infected tissue. Neutrophils promote the recruitment of monocytes via the release of CCL2, while the secretion of CCL3, CCL4 and CCL5 by macrophages results in B and T cell migration. MSCs express and respond to chemokines and to date, the CXCL12-CXCR4 axis is the best described axis in MSC homing to wounds.CXCL12 is a homeostatic chemokine and its cognate receptor is CXCR4. ACKR3 is also able to interact with CXCL12 but this interaction does not result in chemotaxis but it has been described to be important for endothelial cell proliferation , 45. CXCMore importantly, pre-treatment of MSCs with CXCL12 increases their survival and proliferation potential, as well as enhancing their migration towards injured tissue and increasing the secretion of basic fibroblast growth factor and VEGF in vitro . MoreoveFrom the perspective of transplant clinicians or researchers, pre-treatment of cells and tissues is considered more than minimal manipulation and for commercial use, it requires GMP methodologies to ensure that cells meet the requirements for safety, while maintaining the identity, quality and purity characteristics that the cells are represented to possess prior to ex vivo manipulation . For thiMSCs express variable levels of chemokine ligands and receptors but migration of MSCs towards chemokines has only been described in a few cases.The CCL27-CCR10 axis has been shown to recruit CD34 + bone marrow derived multipotent cells towards the skin. CCR10 expression has been described in human and murine MSCs isolated from different sources , 56 and CCR7 expression is widely described in MSCs and CCL19 and CCL21 are the only ligands for CCR7. High expression of CCL21 by secondary lymphoid organs has been described to be responsible for the non-specific distribution of infused MSCs . HoweverThe CCL5-CCR5 axis is involved in the recruitment to wounds of endothelial progenitor cells in vivo  and hemaWithin a clinical context, the optimal delivery method of MSCs should provide the highest regenerative benefit with the lowest side effects. The most used routes of MSC administration, outside tissue-engineering-based methods, are direct injection into the tissue of interest and systemic infusion, both intra-venously (IV) or intra-arterially. Direct injection should have the advantage of a much more precise localization of the cells; however, only 1 to 5% of delivered cells engraft within the target site regardless of the delivery route . Unlike MSCs exert their mechanism via the secretion of paracrine factors that interact with surrounding immune and stromal cells resulting in the generation of pro-tolerogenic, pro-regenerative and anti-inflammatory environments and thus, it is not clear if increased engraftment would result in increased therapeutic potential. Systemic infusion is much less invasive and enables easy access to oxygen and nutrients, which is why it is the preferred method for MSC delivery . SystemiChemokines are master regulators in immune cell trafficking under resting and inflammatory conditions. Specific chemokines have been shown attract immune cells expressing the cognate receptor to specific tissues: the expression of CCR1 in macrophages and neutrophils leads to kidney infiltration in renal ischemia–reperfusion injury ; CCR5 diHuman and murine MSCs have been described to constitutively express chemokines and their receptors, but the expression of these markers varies among reports in the literature due to tissue source of the cells, cell culture methods and passage number of the cells. Murine BM MSCs have been described to express CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR3, CXCR4 and CXCR7 and to have functional CCR3, CCR4, CCR5, CCR7, CCR10 and CXCR4 . AdiposeThere are several strategies to increase the expression of chemokine receptors and improve MSCs homing efficiency, including genetic modifications and surface engineering. Viral transduction and mRNA transfection result in permanent or transient overexpression, respectively, of specific chemokine receptors. Viral transduction of CXCR4 increases homing to the bone marrow in irradiated mice  and enhaWhile cell engineering helps improve understanding of the roles of different molecules expressed or secreted by MSCs, the design of good manufacturing practices for cell-engineering is challenging. For cell engineering, large quantities of concentrated vectors must be produced, and cells must be transduced in different areas to avoid contaminating the transduction area with concentrated vector from the production rooms. If the facility produces multiple vectors, separate aseptic rooms for vector production are essential, as well as stringent decontamination processes and scrutinized personnel flow and use of shared equipment to avoid contaminating different areas within the facility. Cell transduction itself, presents different risks of cross-contamination regardless of the smaller quantities of vector required. If different vectors are being transduced for clinical trials or commercial use, it is essential to establish process control systems to ensure vector isolation when in use of common equipment. Cell engineering requirements for commercial or Phase III clinical use in combination with GMP protocols requiring products to be infused fresh, shortly after processing, result in the design of clinical laboratories that are not cost-effective.ISCT stablished guidelines for the phenotypical identification of MSCs however, phenotype does not necessarily equate with therapeutic potential. The mechanism behind the tissue-building and anti-inflammatory role of MSCs is not fully understood but are partially mediated by chemokine secretion. However, there are few studies aimed at understanding the differences between MSCs isolated from different sources as BM MSCs are the main source of MSCs used in the clinic.Wharton's jelly (WJ) of the umbilical cord holds huge potential as an MSC source, and comparative studies have shown that CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8, strong angiogenic chemokines, are upregulated in WJ MSC compared with BM MSC. In contrast, CXCL12 and CXCL13, which are also potent angiogenic factors, were found to be upregulated in BM MSCs .Transcriptional profiling of identically cultured umbilical cord (UC) derived MSCs and adipose derived MSCs showed that these MSCs differed widely in the expression of anti-inflammatory and angiogenic genes in response to inflammatory stimulation . CXCL1, Differential chemokine transcription levels resulted in a differential therapeutic potential in a diabetic mouse model, where islet co-transplantation with UC MSCs into diabetic mice resulted in a better glycemic regulation compared to islet co-transplantation with identically cultured Ad MSCs .Following a similar standardized approach, low purity islet (LPI) derived MSCs and BM MSCs were compared regarding their immunomodulatory, pro-angiogenic and chemotactic potential and it was observed that they expressed similar transcriptional patterns of chemoattractant and inflammation-modulating molecules, with the exception of CX3CL1, which was transcribed at marginally higher levels by LPI MSCs. Comparison of the immunoregulatory potential of LPI and BM MSCs resulted in identical T cell suppression potential in vitro and no significant differences in the total number of immune cells migrating towards the MSCs when infused into an in vivo migration model .The recruitment and homing of MSCs to sites of injury is essential to contribute to tissue repair, revascularization and regeneration, as well as to dampen inflammation and avoid the activation of the immune system. MSCs isolated from different tissues express different chemokine receptors and thus, tissue source of isolation could dictate MSCs migration potential Fig. . UnderstAnalysis of chemokines at transcription and protein levels have shown that anti-inflammatory and pro-angiogenic phenotypes correlated with positive outcomes in a transplant model . Chemoki


Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor type, claiming the lives of patients within 2 years of diagnosis. The major challenges in treating GBM are largely due to the biological characteristics of the tumor and the brain and pharmacokinetics of many drugs approved for other cancers. These tumors are located in areas that make it difficult to surgically resect without posing major issues and exposure to many drugs and therapies are limited due to the blood-brain barrier (BBB). GBMs also contain cancer stem cells, called GSCs, that have self-renewal and tumor initiating abilities, can secrete angiogenic factors, invade into the normal brain, and are chemoresistant and radioresistant. We found that GSCs have an exclusive dependence on core circadian clock transcription factors, Brain and Muscle ARNT-Like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK). These results suggest the potential for small molecule modulators of the circadian clock as a novel therapy paradigm for GBM treatment following surgical resection to prevent GSC infiltration and reoccurrence of the primary tumor. Here we found that multiple classes of clock compounds (Cryptochrome (CRY) stabilizers, REV-ERB agonists, Casein Kinase 1 (CK1) inhibitors, and Casein Kinase 2 (CK2) inhibitors) have the ability to elongate circadian periods in a clock reporter cell line. They also selectively and potently target patient-derived GSCs that range in sensitivity to temozolomide (TMZ) chemotherapy treatment while having limited effects on control cells both as single agents and in combination with each other. This data provides a platform for further exploration of synergistic effects of combining clock compounds with each other or with current GBM therapies, such as chemotherapy and radiation, with the ultimate goal of developing a clinical model of treatment.


The alterations in thyroid function during recombinant human growth hormone (rhGH) treatment have been reported by many authors since this therapy became widely available for patients with growth hormone deficiency (GHD). Decrease of thyroxine level is the most frequent observation in patients treated with rhGH. This paper presents literature data describing changes in thyroid function related to rhGH therapy and a current explanation of mechanisms involved in this phenomenon. The effect of GH on the hypothalamic-pituitary-thyroid (HPT) axis is dependent on a multilevel regulation beginning from influence on the central axis, thyroid, and extra-thyroidal deiodinases activity as well as the impact on thyroid hormone receptors on the end. Changes in central and peripheral regulation could overlap during rhGH therapy, resulting in central hypothyroidism or an isolated slight deficiency of thyroxine. The regular monitoring of thyroid function is recommended in patients treated with rhGH and the decision of levothyroxine (L-thyroxine) supplementation should be made in the clinical context, taking into account thyroid hormone levels, as well as the chance for satisfactory growth improvement. The alterations in thyroid function during growth hormone (GH) therapy have been reported by many authors since the treatment with recombinant human GH (rhGH) became widely available for GH-deficient patients. A decrease in thyroxine level is the most frequent observation in patients treated with rhGH. This phenomenon was described as early as 1975 [The aim of the paper is to show the literature data describing changes in thyroid function related to GH treatment in patients with growth hormone deficiency (GHD) and to present a current explanation of mechanisms involved in this phenomenon.A systematic search for articles was conducted using MEDLINE and PubMed (until July 2021). The keywords used to search were “thyroid hormones” or “hypothyroidism” and “growth hormone treatment” or “rhGH therapy”. The initial number of papers was 559, and finally we found 30 papers that reported thyroid hormones changes in GHD patients (both children and adults) treated with rhGH  1,2,3,4,28,29,30Unfortunately, the results of the studies are inconsistent and do not strictly indicate the cause of the changes in thyroid hormone levels which had been reported. The main differences between the studies included a large heterogeneity of the patient groups, i.e., healthy individuals , non-GHDIn non-GHD subjects, alterations in thyroid function seemed to be less pronounced than in GHD patients ,35,36,37In the literature, there are only few studies evaluating changes in thyroid function during rhGH therapy in a homogenous group of children with isolated GHD ,25,26,30Most studies on the effects of rhGH therapy on thyroid function included mixed group of patients, both children and adults, with isolated GHD (IGHD) or MPHD, and their results are divergent see . ChangesThe unmasking of central hypothyroidism after the initiation of rhGH therapy has also been reported as a cause of the hypothalamic-pituitary-thyroid (HPT) axis alterations, especially in patients with severe GH deficit coincided with MPHD ,19,27,38The mutual interplay between the GH/IGF-1 axis and the HPT axis is variable. Biological evidence of close relationships between the two axes are mainly based on clinical observations in GH-deficient patients treated with rhGH at physiological doses and also in opposite circumstances—in acromegalic patients with pathological GH excess, in whom the thyroid gland is often affected. In patients with supraphysiological level of GH, goiter is reported in 55–87% ,40 and tJørgensen et al.  postulatStudies in GHD children suggest that the absence of the nocturnal TSH increase could predict the development of hypothyroidism during rhGH replacement . AdditioThe most frequent alteration of thyroid hormone balance in patients under rhGH treatment is decreased fT4 and rT3 with increased fT3. These changes are explained by the influence of GH on the activity of deiodinases . Glynn et al.  investigAn additional mechanism which could be involved in thyroid hormone balance disorders after rhGH therapy is its impact on thyroid hormones receptors. The presence of thyroid hormone receptors (TRs) in growth plates is well known . SusperrConsidering the above, we can summarize that the effect of GH on thyroid hormones is dependent on a multilevel regulation involved influence on the central axis, thyroid, and extra-thyroidal deiodinases activity and thyroid hormone receptors . NumerouThe unsolved issue is the indication for the L-thyroxine substitution in the patients treated with rhGH. Only a few patients develop overt hypothyroidism, but there is clinical evidence of the positive impact of higher thyroxine levels on growth velocity in children treated with rhGH . ThyroxiIn conclusion, we can summarize that rhGH treatment influenced the HPT axis on the central as well as on the peripheral level and changes could appear at any time during therapy. The group with a high risk of hypothyroidism are patients with MPHD or organic GHD, but it should also be considered in patients with a deteriorated growth effect during rhGH therapy. The decision of L-thyroxine supplementation should be made in the clinical context, taking into account thyroid hormone levels as well as a satisfactory growth effect during rhGH treatment.


CKX1/6, EXPA3, MAX4, CYCD3;1, SUSY, 6PFK, APX1, and RBOHB1). Because these genes are affecting branching through different hormonal or metabolic pathways and because expression of some of these genes is photoregulated, our bioinformatic analysis suggests that miRNAs may trigger a rearrangement of the regulatory network to modulate branching in response to light environment.Shoot branching is highly dependent on environmental factors. While many species show some light dependence for branching, the rosebush shows a strict requirement for light to allow branching, making this species an excellent model to further understand how light impinges on branching. Here, in the first part, we provide a review of the current understanding of how light may modulate the complex regulatory network of endogenous factors like hormones , nutrients (sugar and nitrogen), and ROS to control branching. We review the regulatory contribution of microRNAs (miRNAs) to branching in different species, highlighting the action of such evolutionarily conserved factors. We underline some possible pathways by which light may modulate miRNA-dependent regulation of branching. In the second part, we exploit the strict light dependence of rosebush for branching to identify putative miRNAs that could contribute to the photocontrol of branching. For this, we first performed a profiling of the miRNAs expressed in early light-induced rosebush buds and next tested whether they were predicted to target recognized regulators of branching. Thus, we identified seven miRNAs  that could target nine genes ( Important agronomic traits such as yield, visual and sanitary qualities, harvest index and even organoleptic quality are determined by plant architecture in general and shoot branching in particular . A lot oBranching relies on the ability of an axillary bud, a structure containing a miniature shoot comprising a meristem, short internodes and immature leaves, to break dormancy and to grow through cell proliferation and expansion into a new branch. Dormancy is complex and includes endo-, para-, and eco-dormancies that can in part overlap . While eLight signal is particularly informative for the plant since it varies in intensity, quality, direction, and duration . The shaBRANCHED1 (RhBRC1) in interaction with sugars and regulation of its 3′UTR region with the potential role of PUMILIO RNA-BINDING PROTEIN FAMILY 4 (RhPUF4)  .Caenorhabditis elegans  regulation is a major component of post-transcriptional regulation of all biological processes in eukaryotes. First discovered in  elegans , miRNAs entified . MiRNAs entified . Some mientified . Conserventified .per se. Here, we provide a survey of the current knowledge on this subject, first sketching how the main mechanisms regulating axillary bud outgrowth may be connected to light control. Next, we provide a comprehensive view of axillary bud outgrowth regulation by miRNAs and, exploring the wider literature, we provide hypotheses on how light controls miRNAs activity and may contribute to the photocontrol of axillary bud outgrowth. In a second part, based on our expertise in rosebush and using in silico analysis based on the miRNA profiling we performed, we further explored the rose genome to identify and discuss novel miRNAs-gene target couples that may play a potential role in the regulation of axillary bud outgrowth and in its photocontrol in rosebush. Thus, we provide novel hypotheses on the miRNA-mediated regulation of bud growth photocontrol on which future research may stand.Some knowledge has been gained on the roles of miRNAs in meristem initiation  and RoGA3ox and reduction of GA catabolism through down-regulation of RoGA2ox were shown to contribute to axillary bud outgrowth , cytokinins (CK), and strigolactones (SL) form a core regulatory hormonal network instrumental for the release of apical dominance and induction of axillary bud outgrowth . The balG2 phase . ApplicaG2 phase  while beG2 phase . Last, gG2 phase . In roseutgrowth .in vitro experiments showed axillary bud outgrowth is dependent on sucrose availability  and RhSWEET10 control sucrose transport to the bud (RhSUSY and VACUOLAR INVERTASE 1 (RhVI1) catabolize sucrose into fructose and glucose for growth , YUCCA 1 (RhYUC1)] and auxin efflux  genes in buds and represses strigolactones MORE AXILLARY BRANCHES 2 (RwMAX2) signaling gene. Promoting effect of sucrose onto axillary bud outgrowth also relies on repression of a main negative regulator of axillary bud outgrowth BRANCHED1 (BRC1)   by sucro1 (BRC1) .CATALASE (CAT) scavenging activity, causing accumulation of hydrogen peroxide (H2O2) that promotes bud outgrowth , CELL DIVISION CYCLE 2 (CDC2), CYCLIN B  takes for example rapidly place in pea  and blue (B) lights promote axillary bud outgrowth while far-red (FR) light is inhibitory . These rISOPENTENYLTRANSFERASE ], activation [LONELY GUY 8 (RhLOG8)], and transport [PURINE PERMEASE 5 (RhPUP5)] and to the repression of CK catabolism gene CYTOKININ OXIDASE/DEHYDROGENASE 1 (RhCKX1). This leads to CK accumulation in nodes and buds which then causes an up-regulation of IAA biosynthesis and transport , sugar  synthesis and transport genes, and repression of axillary bud outgrowth inhibitory genes . CK upregulation during light control of rosebush axillary bud outgrowth also acts through ROS scavenging  and exogenous hormonal and sugar treatments. From these studies, demonstration was made that CK are initial targets of the light control pathway . More pravenging . In partt in bud . These lPA genes , 2017. APA genes . Light rne GA2ox .RhBRC1 and SL signaling RwMAX2 as well as repression of ROS scavenging AsA-GSH pathway (NAD-DEPENDANT SORBITOL DEHYDROGENASE (RhNAD-SDH) transcriptional activity is indeed observed upon exposure of buds to darkness  has been instrumental in uncovering the role of miRNAs in bud outgrowth. To-date, miR156 is one of the best characterized miRNA families. MiR156 plays a major role in regulating shoot branching by targeting a subset of the SQUAMOSA PROTEIN-LIKE (SPLs) genes. SPL transcription factors are found in all green plants and are involved in many developmental regulatory processes such as phase transition and flowering, plastochron control, leaf development, fruit ripening and response to stresses  that improves rice productivity. On the contrary, high miR156 levels increase tiller formation in the dominant Corngrass1 maize mutant  and AUXIN SIGNALING F-BOX 2 (OsAFB2)]. Accordingly, rice lines overexpressing miR393 show a reduced response to auxin and lower expression of the OsTB1 gene, which may account for increased axillary bud growth  that develop less tillers  or HAIRY MERISTEM (HAM)] ] . These pM (HAM)] . ArabidoAM genes . In toma species . SlGRAS2nown yet . The funnown yet .CUP-SHAPED COTYLEDON (CUC) genes that are important for a wide range of meristem-related developmental processes. Recent studies performed in cotton and in the heterologous Arabidopsis system suggest that repression of CUC2 by miR164 is important for branch outgrowth , overexpression of miR319 leads to a slight decrease in tillering (TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor family which are involved among others in the transition from cell proliferation to differentiation during plant development . Similar numbers of members were found in Arabidopsis except for miR171, 172, and 319 families in which a much lower number of members were identified in Arabidopsis  in comparison to Rosa. This may suggest differences in the tuning of the regulations controlled by these miRNA families between Rosa and Arabidopsis.Our bioinformatic analysis shows that out of the 9 miRNAs families previously described as involved in the control of bud outgrowth (as mentioned above), 7 were found in Rosa . The twoRosa . Each RoRosa miRNAs to known targets was predicted by searching sequences complementary to the miRNAs in the putative targets. Same target gene families were found in the Rosa genome as described for other species  and Blue (B) light signal transduction engages photoreceptors, including phytochromes (PHY) and cryptochromes (CRY) and their downstream effectors as ELONGATED HYPOCOTYL 5 (HY5) and PHYTOCHROME INTERACTING FACTORS (PIF4). PHYB plays a main role in the photocontrol of bud outgrowth (phyB loss-of-function and a low R/FR ratio. This phenotype is correlated to BRC1 expression induction by PIF4/PIF5 and to abscisic acid (ABA) accumulation in axillary buds. Interestingly, several reports indicated that modulation of miRNA expression may contribute to the PHY-mediated light response, raising the hypothesis that this may contribute to branching modulation. For instance, analysis of miRNAs and their PHYB-mediated targets in rice leaves identified a total of 135 miRNAs differentially expressed between the WT and phyB mutant  both targeted by miR159a_1 and one in the strigolactones pathway (RwMAX4) targeted by six miR166 members. These genes are involved in the repression of bud outgrowth in Rosa. Interestingly, none of the genes involved in auxin and gibberellin pathways are found as potential targets of post-transcriptional regulation via miRNAs. Concerning sugar control, two genes  are targeted by two different miRNAs: miR8175 and miR166e-5p, respectively. Besides hormonal and sugar control, two genes involved in ROS metabolism are potential targets of miRNAs. The first one, RhAPX1, encoding antioxidant enzyme is targeted by three miR164 isoforms , the other one (RhRBOHB1) encoding a NADPH oxidase is targeted by miR399e_5. Finally, two genes involved in the cell division  and expansion (RhEXPA3) are identified as potential targets of miR477e and miR156j_1, respectively. The validation of the interaction between these 7 miRNAs and the predicted rosebush targets awaits experimental confirmation. It is however noteworthy that, in agreement with our findings, miR159 was previously shown as targeting CKX with negative correlation of their expression in response to pathogens in poplar  lack a binding site for the miRNA (data not shown). Therefore, it is attractive to speculate that the addition of these new targets may have appeared by evolution of their sequence to create a novel miRNA binding site. The resulting bifurcated pathway downstream the miRNA, with one conserved branch (SPL or NAC) and one branch more species-specific (EXPA3 or APX1) could provide robustness to the effect of the miRNA on bud outgrowth.The other two miRNAs identified here, miR156 and miR164 were already described to play a role in regulating shoot branching (as mentioned above). While miR156j_1  and NAC , new tar in Rosa . The AraRosa we explored specifically the potential role of miRNAs in the control of bud growth and how they may manage to interact with this network. Thus, we identified through bioinformatic analysis some conserved miRNAs families and their associated target genes as good candidates to play a potential role in the control of axillary bud outgrowth in the rosebush. Interestingly, these miRNAs have the ability to target key molecular players in all major pathways regulating bud outgrowth  with a far broader spectrum compared to previously reported studies. Additionally, according to literature, several of those such as miR156, miR164, miR166, and miR8175 are under light regulation which leads us to suggest that they may participate in the light-mediated mechanisms of axillary bud outgrowth. Whether or not these novel miRNA-gene target couples physically interact and demonstrate a biological relevance in the regulation of bud outgrowth and its photocontrol is still to be addressed. To this end, we need to move toward functional analysis and use of mutants.Multiple regulators have been described as acting in concert to fine tune, in an intricate network, axillary bud outgrowth as well as its photoregulation. While this network is mostly built on integrating knowledge gained at the transcriptional level, little is known to date about how post-transcriptional regulation fits into this network. Here, we provide new information by integrating post-transcriptional regulation of the photocontrol bud growth into this network. Based on the literature published mainly on Arabidopsis, rice and maize combined with our bioinformatic analysis on Involvement of post-transcriptional factors in bud outgrowth control is not limited to miRNAs. Indeed, some studies have brought to light the possible role of small interfering RNAs (siRNAs) in this process . In thisThe understanding of the mechanisms behind miRNA control of bud outgrowth could be a great opportunity for improving agriculturally important traits such as plant architecture . NotablyThe original contributions presented in the study are included in the article/JM, PL, NL, and JLG designed the experiments and wrote the manuscript. JM performed the experiments and bio informatic analysis. PL, NL, and JLG supervised the work. All authors read and approved the final 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.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.


The global measles vaccination program has been extraordinarily successful in reducing measles-related disease and deaths worldwide. Eradication of measles is feasible because of several key attributes, including humans as the only reservoir for the virus, broad access to diagnostic tools that can rapidly detect measles-infectious persons, and availability of highly safe and effective measles-containing vaccines (MCVs). All 6 World Health Organization (WHO) regions have established measles elimination goals. Globally, during 2000–2018, measles incidence decreased by 66% (from 145 to 49 cases per million population) and deaths decreased by 73% (from 535 600 to 142 300), drastically reducing global disease burden. Routine immunization with MCV has been the cornerstone for the control and prevention of measles. Two doses of MCV are 97% effective in preventing measles, qualifying MCV as one of the most effective vaccines ever developed. Mild adverse events occur in <20% of recipients and serious adverse events are extremely rare. The economic benefits of measles vaccination are highlighted by an overall return on investment of 58 times the cost of the vaccine, supply chains, and vaccination. Because measles is one of the most contagious human diseases, maintenance of high (≥95%) 2-dose MCV coverage is crucial for controlling the spread of measles and successfully reaching measles elimination; however, the plateauing of global MCV coverage for nearly a decade and the global measles resurgence during 2018–2019 demonstrate that much work remains. Global commitments to increase community access to and demand for immunizations, strengthen national and regional partnerships for building public health infrastructure, and implement innovations that can overcome access barriers and enhance vaccine confidence, are essential to achieve a world free of measles. Measles is a febrile rash illness that can lead to serious complications and death and one of the world’s most contagious viral diseases. The basic reproduction number (or average number of secondary cases generated by an infectious person in a fully susceptible population) for measles is estimated to be 12–18, higher than that of many other common childhood illness . Under the assumption of a homogenously mixing population, such high transmissibility means that significantly high population immunity levels of >92%–94% are needed to impede sustained measles virus transmission. Measles vaccines have been enormously successful in controlling measles globally, demonstrating the feasibility of reaching a measles eradication goal . In the current article, we review the significant impact measles vaccination uptake has had on reducing measles disease burden worldwide, the origin of measles vaccines and their safety and effectiveness profiles, global trends in vaccination coverage, the economic benefits of investing in measles vaccination, the various setbacks encountered in measles control in recent years, and key challenges that must be overcome to achieve a world free of measles.Before the introduction of measles vaccination, measles caused substantial human disease and death worldwide, infecting nearly everyone by 15 years of age. Measles was common in all parts of the world and caused an estimated 135 million cases and more than 6 million deaths globally each year [1]. In the United States alone, an estimated 3–4 million people acquired measles every year , of which approximately 500 000 cases and nearly 500 deaths were reported annually [2].Although most persons fully recover from measles without sequelae, the disease entails significant morbidity and mortality risks. Common complications of measles include otitis media and diarrhea, but more serious complications can also occur and include pneumonia, encephalitis, and subacute sclerosing panencephalitis, a slowly progressive neurologic sequela of measles that is universally fatal  . SecondaAfter more widespread use of measles vaccines globally in the 1980s, measles incidence and mortality rates decreased to low levels in all regions of the world [11]. During 2000–2018, the worldwide annual reported measles incidence per million population decreased by 66%, from 145 to 49 cases, the annual number of reported measles cases decreased by 59%, from 853 479 to 353 236 , and annAs of 2019, a total of 83 countries have verified measles elimination, paving a path forward for global eradication. Several key attributes position measles as a prime candidate for eradication: (1) absence of a nonhuman reservoir , (2) availability of practical and sensitive diagnostic tools, such as immunoglobulin (Ig) M serologic assays and molecular tests to identify persons with acute measles, and, most importantly, (3) highly effective measles vaccines. Measles eradication is feasible ; howeverMeasles virus is a negative sense, single-stranded RNA virus with a genome size of 15 894 nucleotides [16]. Measles virus is considered monotypic ; however, multiple genetically distinct lineages of wild-type measles virus have been described [17]; the WHO currently recognizes 24 genotypes [18].Measles is characterized by a generalized maculopapular skin rash, fever above 38.3°C (101°F), and cough, coryza, and/or conjunctivitis. During the incubation period, the virus replicates in alveolar macrophages and dendritic cells before establishing a systemic infection in which infected lymphocytes disseminate the virus to major organ systems, peripheral tissues, including the skin, the respiratory tract and epithelial cells. In uncomplicated measles cases, clinical signs start to subside a few days after rash onset, and patients develop a robust immune response that mediates recovery and provides lifelong immunity [16].Measles vaccines are attenuated (or weakened) measles viruses, so that infection through vaccination of immunocompetent persons leads to replication and immunity, but not disease. Data suggest that genotype A was widely distributed in the prevaccine era when the progenitors of the vaccine strains were first isolated; however, only a few measles viruses from the 1950s and 1960s have been available for sequencing. All live attenuated vaccine strains used in measles vaccines are members of genotype A.Most of the measles vaccine strains currently in use were derived from the prototype Edmonston strain (Edmonston wild-type) which was isolated by Enders and Peebles in 1954 [19]. In addition to the live attenuated vaccine strains, a formalin-inactivated Edmonston vaccine was in use during 1963–1967; however, use of this vaccine was discontinued because vaccinated individuals were at risk for developing atypical measles after wild-type measles virus infection. Atypical measles is caused by antigen–antibody immune complex deposition and characterized by high fever, abdominal pain, myalgias, pneumonitis, and a petechial or vesicular and edematous rash. The syndrome is preventable by vaccination with a live-attenuated vaccine [20]. To develop live-attenuated vaccines, the Edmonston strain was passaged in human amnion and human kidney cells before being adapted to chicken embryo fibroblasts to generate the commonly used Moraten and Schwarz strains. Several vaccine strains were derived from other wild-type measles virus isolates, including the Leningrad-16 strain used in Russia, the Shanghai-191 strain used in China, and the CAM-70 strain used in multiple countries. All measles vaccines are produced in chicken embryo fibroblasts, except the Edmonston-Zagreb strain (from the Serum Institute of India), which was derived by further passage in human diploid cells, MRC-5 [20].Comparison of the genomic sequences of nine measles vaccine strains with the sequence of the Edmonston wild-type virus has shown a relatively small amount of genetic heterogeneity. Although nucleotide substitutions were found in the noncoding and protein coding regions of the genomes, the overall genetic organization of the vaccine strains was conserved [21], and there are no clear genetic markers for attenuation. The biologic basis for attenuation may be the result of use of different cellular receptors by vaccine and wild-type viruses. Vaccines strains can use both CD46 and human signaling lymphocyte marker for entry, while wild-type viruses recognize only human signaling lymphocyte marker. Vaccine and wild type viruses use nectin 4 to infect epithelial cells before viral shedding . Importantly, the high attenuation of measles vaccine viruses impedes human-to-human transmission of these viruses; a systematic review of 773 articles including genotyping of virus strains in close contacts of vaccinated individuals found that all cases of measles in close contacts were due to wild-type virus [23].The only wild-type measles viruses currently detected in circulation are members of genotypes D8, D4, B3, and H1. Although some antigenic variation between wild-type and vaccine strains has been described [24], the necessity for conservation of the receptor-binding domains on the viral hemagglutinin (H) protein, the surface glycoprotein that is the target of neutralizing antibodies, constrains antigenic drift . This conservation of the H protein allows measles vaccines to be highly effective against all wild-type viruses, directly contributing to overall success of the global measles vaccination program, and likely cessation of transmission of 20 of the 24 known genotypes [25].Vaccination elicits long-lived humoral and cellular immune responses. Serologic correlates for protection from measles virus have been established and a titer >120 mUI/mL is considered protective . Most vaThe safety of MCVs, including measles-only, measles-rubella (MR), measles-mumps-rubella (MMR), and MMR-varicella (MMRV) vaccines is well-established. MCVs have similar safety profiles and are well tolerated, and common reactions after vaccination are mild 20, . Common MMR vaccination is only rarely associated with serious adverse events, and both precautions and contraindications to MMR vaccination have been carefully delineated to minimize serious reactions. A review by the Institute of Medicine assessed whether there was both epidemiologic and mechanistic evidence of serious adverse events associated with MMR vaccine . This reThe attributable risk of febrile seizures after MMR vaccine has been evaluated in multiple large population-based studies to be approximately 25–34 additional febrile seizures per 100 000 children vaccinated, and occurs most commonly 7–14 days after vaccination [35]. The risk is approximately twice as high for children aged 12–23 months who receive the MMRV combination vaccine compared with those who receive MMR and varicella vaccines separately [36]. These studies found that there was 1 additional febrile seizure per every 2300–2600 MMRV vaccine doses given, compared with MMR and varicella vaccines given separately. There was no increased risk in individuals receiving a second dose of MMRV compared to MMR and varicella separately at the recommended 4–6-year-old age range [37]. Based on these findings, providers are recommended to discuss the benefits and risks of vaccination with MMRV versus MMR and varicella separately for the first dose of measles vaccine, and to administer MMRV as the first dose only if the parent or caregiver expresses a preference for MMRV vaccine .Thrombocytopenia can occur after natural measles infection, and a causal relationship between MMR vaccine and thrombocytopenia has been established based on observational studies, case reports, and biologic plausibility . A largeAnaphylaxis and other immediate hypersensitivity reactions can occur after MMR vaccination, and are likely related to allergies to the gelatin or neomycin components of the vaccine [40–42]. In a large population-based study, the risk of anaphylaxis following MMR vaccine was estimated to be 5.14 per million vaccine doses  [43]. History of severe allergic reaction to any component of the vaccine is a contraindication to MMR vaccination .Immunocompromised patients should also not receive the MMR vaccine. Potential fatal adverse events in immunocompromised hosts include measles pneumonia, MIBE, and disseminated measles infection [44–47]. This recommendation is inclusive of persons with human immunodeficiency virus (HIV) infection who are severely immunocompromised. However, a systematic review of 28 safety studies of MMR vaccination among HIV-infected children found that adverse events and deaths after measles vaccines were uncommon in this population [48]; thus, persons with HIV infection should receive MMR vaccine if they are not severely immunosuppressed  .The effectiveness of the measles component of the MMR vaccine is high, 93% after 1 dose and 97% after 2 doses in persons aged ≥12 months. Duration of immunity is likely lifelong after 2 doses . AlthougTiming and delivery strategies for MCV vaccination vary by country and are contingent on multiple factors, including the infrastructure to implement routine immunizations and mass vaccination campaigns, the capacity and preparedness for rapid outbreak response, and the local epidemiology of measles based on surveillance data. Overall disease burden and age at highest risk of disease is often a primary consideration when establishing an immunization schedule. Currently, 41% of countries with measles vaccination programs begin vaccinating infants before 1 year of age , owing to the high disease burden in infants.The chance of exposure to measles for infants and young children in each country must be carefully weighed against the age-specific immunogenicity of each dose of measles vaccine in order to define the optimal age for routine vaccination. Although infants are at risk of severe disease and serious complications, studies have suggested that vaccination at <12 months of age can lead to suboptimal immune responses that continue to be low despite additional doses , 53, 54.During 2000–2019, estimated coverage with the routine first-dose MCV (MCV1) increased globally from 72% to 85% , and theCountries that have achieved measles elimination have invested heavily in routine immunization programs to ensure consistently high vaccination coverage against measles and other vaccine-preventable diseases. In the United States, after the first licensure of measles vaccine in 1963 and MMR in 1971, MCV coverage steadily increased, with almost 20 million doses distributed in 1989 alone [20]. National vaccination coverage surveys including MMR were implemented beginning in 1994 for young children and 2006 for adolescents; estimates have remained steady at ≥90% for both 1 and 2 doses since 2010 . In 2017The economic benefits of investing in vaccines, particularly measles vaccines, are well established 60–64]. Even in countries with low disease incidence, periodic measles outbreaks continue to occur, causing costly disruptions to society and requiring resource-intensive outbreak response activities [65]. A review of cost estimates of 11 measles outbreaks during the postelimination era in the United States, found that measles costs public health and healthcare institutions a median of approximately $33 000 (US dollars) per case, and $4000 per day of investigation . Further–64. EvenDespite significant progress in decreasing measles incidence and mortality rates globally since 2000, measles elimination efforts have encountered setbacks in recent years. Estimated MCV1 coverage worldwide has plateaued at 84%–85% for nearly a decade and reported global measles cases increased from a historic low of 132 490 in 2016 to 869 770 in 2019 (556% increase) , Minnesota [71], and Washington [72]. During 2018 and 2019, prolonged outbreaks of almost 1-year duration in undervaccinated communities in New York threatened the measles elimination status of the United States. The 1282 measles cases reported in the United States in 2019 was the highest annual number of reported cases since 1992 73, . Since 2The Measles & Rubella Initiative (M&RI), a global partnership formed in 2001, coordinates efforts to achieve a world without measles and rubella. The M&RI is led by the American Red Cross, the United Nations Foundation, WHO, United Nations Children’s Fund (UNICEF), and the US Centers for Disease Control and Prevention. Since 2001, it has invested >$1.2 billion for measles and rubella elimination efforts. In 2012, a Measles and Rubella Strategic Plan was released covering the period 2012–2020 and was endorsed by the M&RI [75]. Among its goals were reaching measles elimination in ≥5 of the 6 WHO regions, establishing a target date for measles eradication, and achieving a ≥95% coverage with MCV in all districts of all countries no later than 2020. While considerable progress in measles control has been made, none of those targets have been met.There are multiple challenges to achieving and maintaining the measles herd immunity threshold needed for measles eradication, estimated generally to be >92%–94%. Achieving such immunity levels requires substantial political will, elimination of financial and physical access barriers to measles vaccination, strengthening of public health infrastructure , and combating dissemination of misinformation eroding trust and confidence in vaccines . A midterm review of the Measles and Rubella Strategic Plan concluded that measles could be eradicated and offered a number of recommendations to try to support eradication . NotablyIntensified efforts and resource commitments by global partners and countries are needed to get back on track toward measles elimination. A new immunizations guidance document, the Immunization Agenda 2030 (IA2030) [82], cocreated by WHO and partners and to be endorsed by the World Health Assembly, builds on lessons learned and progress made toward the Global Vaccine Action Plan goals. The IA2030 aims to use measles, a proven effective surrogate or marker for the performance of Expanded Programmes on Immunization [83], to drive efforts to strengthen immunizations and primary healthcare systems [82].Focusing on measles elimination strategies can enhance delivery of routine immunization for other vaccine-preventable diseases, help identify unvaccinated or undervaccinated communities and close immunity gaps, lead to improvements in surveillance and expansion of cold-chain capacity, create opportunities to provide refresher training on vaccination to healthcare workers, and advance the adoption of strategies used for measles elimination to ensure high coverage for vaccines against other diseases  [84]. The IA2030 thus provides an opportunity to strengthen vaccination programs, build on public health partnerships, and leverage data-driven approaches that use disease surveillance to increase vaccination coverage and equity in all communities .Innovative approaches for measles diagnostics and vaccination methods, principally to address challenges in resource-limited settings lacking strong healthcare infrastructures, could facilitate the control of measles and help overcome critical barriers to achieving measles elimination.Because many of the typical clinical signs of measles can also be caused by other infectious agents, including rubella virus, laboratory confirmation of measles is a critical component of the measles control strategy but may not always be feasible in resource-limited settings. Detection of measles-specific IgM antibodies by enzyme immunoassay is the most common method used for case confirmation, though detection of viral RNA by reverse-transcription polymerase chain reaction is increasing in many countries. While most of the IgM tests are performed with serum samples, the use of alternative samples, such as dried blood spots and oral fluid samples, has helped to expand laboratory surveillance. The recent development of a rapid diagnostic test that can provide results in <20 minutes to detect measles IgM in field settings, will facilitate rapid detection of cases and response activities in resource-limited settings where the logistics of sample transport and storage are often challenging 86, .Alternative vaccine delivery methods that eliminate the need for cold-chain transportation and hypodermic needle and syringe subcutaneous injection could improve vaccine delivery in resource-limited settings and increase vaccination coverage and equity. Several routes of administration have been evaluated. A measles vaccine delivered by the respiratory route as an aerosol was immunogenic but the seroconversion rate was inferior to the rate observed after subcutaneous injection [88]. A dry powder was immunogenic in nonhuman primates, but work was discontinued after a phase I clinical trial . Measles vaccination via the intradermal route produced lower seroresponses because efficient and reliable delivery methods were not available [91]. However, the recent success in development and testing of dissolving microneedle patches for the delivery of measles and rubella vaccine shows that intradermal vaccination by this method is a promising way to improve the efficiency of delivery of vaccination 92, .Although measles is one of only a handful of human pathogens that could be eradicated, and despite the broad availability of an intervention tool that could make this possible—an effective and inexpensive measles vaccine with decades of use and an excellent safety track record—the disease remains a leading cause of childhood disease and death globally. Several tough challenges to increasing measles vaccination coverage remain, including poor access to immunization services and dissemination of misinformation eroding vaccine confidence.Exacerbating these challenges are the recent diversions of public health resources to combat the novel coronavirus disease 2019 pandemic, which have affected routine immunization services and vaccine-preventable disease surveillance in many countries, thereby increasing the risk for outbreaks of measles during or after the pandemic. Yet, the notable successes of measles vaccination in the control and elimination of measles in various regions across the globe undoubtedly demonstrate that these barriers can be overcome, and that measles eradication is attainable. With renewed efforts and resource commitments to achieve elimination, and the implementation of strategies that improve primary health delivery systems, decrease missed opportunities for vaccination and counteract vaccine hesitancy, and incorporate innovative approaches for measles diagnostics and vaccine delivery, a world without measles could be within our grasp.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at The complete references are available as online Supplemental Material.jiaa793_suppl_Supplementary-MaterialClick here for additional data file.