Six fresh frozen human thoracic spine specimens with intact rib cages (C7-L1) were loaded with pure moments of 5 Nm in flexion/extension, lateral bending, and axial rotation, while monitoring the relative motions of all spinal segments using optical motion tracking. The specimens were tested (1) in the intact condition, followed by testing after vertebral body replacement at T6 level using a unilateral approach combined with (2) long instrumentation (T4–T8) and (3) short instrumentation (T5–T7) as well as a bilateral approach combined with (4) long and (5) short instrumentation. Significant increases of the range of motion (p < 0.05) were found in the entire thoracic spine (T1–T12) using the bilateral approach and short instrumentation in primary flexion/extension and in secondary axial rotation during primary lateral bending compared to both conditions with long instrumentation, as well as in secondary lateral bending during primary axial rotation compared to unilateral approach and long instrumentation. Compared to the intact condition, the range of motion was significantly decreased using unilateral approach and long instrumentation in flexion extension and secondary lateral bending during primary axial rotation, as well as using bilateral approach and long instrumentation in lateral bending. On the segmental level, the range of motion was significantly increased at T4–T5 level in lateral bending using unilateral approach and short instrumentation and significantly decreased using bilateral approach and long instrumentation compared to their respective previous conditions. Regardless of the approach type, which did not affect thoracic spinal stability in the present study, short instrumentation overall shows sufficient primary stability in the mid-thoracic spine with intact rib cage, while creating considerably more instability compared to long instrumentation, potentially being of importance regarding long-term implant failure. Moreover, short instrumentation could affect adjacent segment disease due to increased motion at the upper segmental level.Spinal tumors and unstable vertebral body fractures usually require surgical treatment including vertebral body replacement. Regarding primary stability, however, the best possible treatment depends on the spinal region. The purpose of this  Combined posterior fusion using pedicle screw-rod systems and anterior spinal support using vertebral body replacement implants represents the most common treatment in unstable vertebral body fractures . FurtherPrevious investigations showed that most vertebral body fractures occur in the thoracolumbar region, more specifically at the T12 and L1 levels . Althougin vitro study therefore was to evaluate the effects of (1) the length of instrumentation (one level above/one level below vs. two levels above/two levels below) and (2) the type of surgical approach  on thoracic spinal stability (defined as primary ranges of motion) and motion behavior (defined as secondary ranges of motion) when performing vertebral body replacement combined with posterior fusion and considering the rib cage.The stability and motion behavior of combined vertebral body replacement and posterior spinal fusion in terms of the primary and secondary ranges of motion have mainly been investigated on the lumbar spine in the past. However, due to the stabilizing effect of the rib cage and a diverging spinal morphology, the optimum treatment for the thoracic spine may differ from that for the lumbar spine. The purpose of this 3, ranging from 2 to 96 mgHA/cm3 (Six fresh frozen human thoracic spine specimens (C7-L1), including the intact rib cage, were gathered for biomechanical testing. Four of the six specimens originated from male and two from female donors, together exhibiting a mean age of 81 ± 13 years, ranging from 63 to 99 years, as well as a mean bone mineral density of 38 ± 35 mgHA/cmmgHA/cm3 . Prior tin vitro testing of spinal implants . In the first step, the specimens were loaded in the intact condition without surgical approach or posterior instrumentation , 2-1. InRanges of motion and neutral zones of the entire thoracic spine (T1–T12) as well as the two adjacent levels above and below the vertebral body replacement  were evaluated from the hysteresis curves of the third full loading cycles in all primary and secondary motion planes using Matlab 2017  and post-processed using Excel 2016 . All primary (in-plane) and secondary (out-of-plane/coupled) ranges of motion were grouped and statistically analyzed for significant differences using Friedman ANOVA with Bonferroni correction and a significance level of 0.05. Statistical analysis was performed using SPSS 24 . Grouped data were represented and visualized as median values with respective value ranges, while negative values characterized inverse motions, for instance, extension during primary flexion and vice versa.in vitro study was approved by the ethical committee board of the University of Ulm, Germany, in April 2018 (No. 487/17). The specimens were acquired from the anatomy department of the University Hospital Aachen, Germany, in November 2018, which declared that written informed consent of the donors was obtained prior to decease. The study was funded by the German Research Foundation  and the Kazakhstan Ministry of Public Health (32-18-280/129). The authors declare to have no potential conflicts of interest.The use of human specimens in the present Compared to the intact condition, no significant increases of the primary and secondary ranges of motion of the entire thoracic spine (T1–T12) were found for all testing steps –5. In prConsiderable effects of the instrumentation length were found in both primary lateral bending  and primOn the segmental level, no significant range of motion value changes were found, neither in primary nor in secondary motion planes, except for the primary lateral bending range of motion at the T4–T5 level . Here, uAll results are given in summarized form in the Vertebral body replacement combined with posterior instrumentation using pedicle screw-rod constructs is widely used in the surgical treatment of spinal diseases such as unstable vertebral body fractures, spinal tumors, and other pathologies. Regarding primary stability, however, the length of instrumentation and the type of surgical approach depends on several factors, such as the spinal morphology or the effect of stabilizing ligamentous and bony structures. Since these parameters were mainly studied in the lumbar spine in the past, the present biomechanical study aimed to evaluate the effects of long and short instrumentation as well as the effects of uni- and bilateral surgical approaches on the primary stability of the thoracic spine, including all rib cage structures to represent a more physiological condition.The findings of the present study generally revealed that thoracic spinal stability is maintained after vertebral body replacement, independent of instrumentation length or surgical approach type, when compared to the intact condition where no vertebral body replacement implant and no posterior instrumentation were used. However, short instrumentation (one level above/one level below) was found to create distinctly more range of motion compared to long instrumentation (two levels above/two levels below), especially in primary flexion/extension using the bilateral approach type. Furthermore, short instrumentation altered the coupled motion characteristics of the entire thoracic spine (T1–T12) in both primary lateral bending and primary axial rotation, leading to increased secondary axial rotation and secondary lateral bending, respectively, when using the bilateral approach type. These findings might indicate that short instrumentation more likely induces long-term implant failure or pedicle screw loosening, assuming that higher flexibility is related to higher stresses in the rods and the vertebral bone of the adjacent segments. Moreover, short instrumentation led to a significant range of motion increase at the segmental level T4–T5 in primary lateral bending using the unilateral approach type compared to long instrumentation, indicating a higher risk of adjacent segment degeneration when hypothesizing that increased range of motion in the respective motion segment is a primary risk factor for adjacent segment disease. Furthermore, variation ranges were detected to be larger after posterior instrumentation compared to the intact condition at all adjacent segmental levels and in all motion planes, indicating varying effects of the posterior instrumentation regarding level and motion direction. Regarding the surgical approach type, no clear indications were found that the bilateral surgical approach causes higher instability compared to the unilateral approach when considering comparisons between the long and short instrumentation groups, respectively. Combined vertebral body replacement and posterior fusion therefore seems to compensate the destabilization induced by the removal of all ligamentous and bony structures due to the surgical approach.in vitro studies  may be generally recommended after vertebral body replacement in the thoracic spine to avoid the risks of implant failure, pedicle screw loosening, or adjacent segment disease. Short instrumentation (one level above/one level below), however, also ensures reasonable primary stability and thus is an acceptable alternative in individual cases, for instance, when the extent of surgical approach has to be reduced for medical reasons. The bilateral surgical approach type, as used for the treatment of vertebral tumors, represents a secure surgical procedure from a biomechanical point of view when combined with vertebral body replacement implant and posterior fusion. Nevertheless, the bilateral surgical approach entails a major destabilizing surgical intervention which should be treated with at least long posterior instrumentation.All datasets generated for this study are included in the article/in vitro study was approved by the ethical committee board of the University of Ulm, Germany, in April 2018 (No. 487/17). The specimens were acquired from the anatomy department of the University Hospital Aachen, Germany, in November 2018, which declared that written informed consent of the donors was obtained prior to decease.The use of human specimens in the present CL contributed to study design, experimental testing, data analysis, and manuscript preparation. TKo contributed to experimental testing, discussion, and manuscript review. VA contributed to funding acquisition, experimental testing, discussion, and manuscript review. TKe contributed to funding acquisition and manuscript review. SA contributed to funding acquisition and manuscript review. JJ contributed to experimental testing, data analysis, and manuscript review. MV contributed to experimental testing, data analysis, and manuscript review. H-JW contributed to funding acquisition, discussion, and manuscript review.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.


In utero patency of the ductus is maintained by low oxygen tension, high levels of prostaglandins, nitric oxide and carbon monoxide. After birth, ductal closure occurs first by functional closure, followed by anatomical remodeling. High oxygen tension and decreased prostaglandin levels mediated by numerous factors including potassium channels, endothelin-1, isoprostanes lead to the contraction of the ductus. Bradykinin and corticosteroids also induce ductal constriction by attenuating the sensitivity of the ductus to PGE2. Smooth muscle cells of the ductus can sense oxygen through a mitochondrial network by the role of Rho-kinase pathway which ends up with increased intracellular calcium levels and contraction of myosin light chains. Anatomical closure of the ductus is also complex with various mechanisms such as migration and proliferation of smooth muscle cells, extracellular matrix production, endothelial cell proliferation which mediate cushion formation with the interaction of blood cells. Regulation of vessel walls is affected by retinoic acid, TGF-β1, notch signaling, hyaluronan, fibronectin, chondroitin sulfate, elastin, and vascular endothelial cell growth factor (VEGF). Formation of the platelet plug facilitates luminal remodeling by the obstruction of the constricted ductal lumen. Vasa vasorum are more pronounced in the term ductus but are less active in the preterm ductus. More than 100 genes are effective in the prostaglandin pathway or in vascular smooth muscle development and structure may affect the patency of ductus. Hemodynamic changes after birth including fluid load and flow characteristics as well as shear forces within the ductus also stimulate closure. Current pharmacological treatment for the closure of a patent ductus is based on the blockage of the prostaglandin pathway mainly through COX or POX inhibition, albeit with some limitations and side effects. Further research for new agents aiming ductal closure should focus on a clear understanding of vascular biology of the ductus.Failure of ductus arteriosus closure after preterm birth is associated with significant morbidities. Ductal closure requires and is regulated by a complex interplay of molecular and mechanical mechanisms with underlying genetic factors.  Fetal circulation is a unique event, functionally different from pediatric and adult circulation. Ductus arteriosus (DA) is a small, simple vessel with just a few milimeters length and width, but has a profound impact on the survival of the fetus and newborn and plays an essential role in normal postnatal adaptation after birth. It functions as a bridge between two large vessels in fetal life, but sometimes it becomes the “bridge between life and death” in preterm infants. It has baffled scientists since ancient times, and beginning from Galen and Ibn-al Nafis, Vesalius, Leonardo Botallo and William Harvey have commented on its structure and function .During fetal development, cardiovascular septation occurs during days 22–28 of gestation and looping of ventricles completes. At this stage, bilateral aortic arches and bilateral DA form but over the next week, the right aortic arch and ductus involutes. Ductus arteriosus arises from the left sixth embryonic arch; leaves the pulmonary artery very close to the bifurcation point and inserts at the transition zone between the aortic arch and descending aorta, distal to the origin of the left subclavian artery. Usually it has a tubular shape but may be funnel-shaped with a narrow end on the pulmonary side and a wide end on the aortic side  and the anatomical step (remodeling). Interestingly, the closure of DA resembles the process after vascular injury or atherosclerosis in the adult arteries . UnderstSmooth muscle tone is regulated by phosphorylation/dephosphorylation of myosin light chain (MLC). MLC is phosphorylated by Calcium/calmodulin-dependent MLC-kinase (MLCK) and dephosphorylated by calcium independent MLC-phosphate (MLCP). Increased cytosolic calcium activates MLCK, which leads to MLC phosphorylation and vasoconstriction  Figure . PatencyProstaglandins are synthesized from arachnidonic acid by cyclooxygenases COX1 and COX2 within the ductal muscle. Circulating prostaglandins, originated from the placenta contribute to the patency of DA . Prostag2) is the major arachnidonic acid product of the ductus but although it is more prevalent, it is less potent than prostaglandin E2 (PGE2), which is the most important prostaglandin to regulate the patency of DA (2 interacts mainly with its receptor EP4. EP4 activates voltage-gated potassium channel (Kv) and outward K+ current increases, thereby inducing membrane hyperpolarization, which inhibits Ca2+ influx via the voltage-gated L-type calcium channels (CaL) and decreases intracellular calcium , which inturn activates protein kinase (PKA), which inhibits myosin light chain creatine kinase (MLCK). Inhibition of MLCK blocks phosphorylation of myosin light chains, which results in inhibition of vasoconstriction, hence vasodilation occurs , by PKG leads to uptake of Ca2+ in sarcoplasmic reticulum, lowering intracellular calcium  is produced by endothelial nitric oxide synthase in the luminal endothelium and endothelium of vasa vasorum and maintains the patency of DA by acting through cGMP. After its production, it diffuses into the SMC and binds with soluble guanylyl cyclase, producing cyclic guanosine monophosphate (cGMP). cGMP activates cGMP-dependent protein kinase (PKG), which induces vasodilation  Figure . NO is ather one . This mi2 sensing cytochrome P450, thus interrupting endothelin-1 signaling (Hem oxygenase 1 and hem oxygenase 2 which produce carbon monoxide (CO) are found in the endothelial and smooth muscle cells of the DA. CO dilates the ductus by inhibition of Oignaling , 20  inhibitors such as milrinone and amrinone have positive inotrope and vasodilator effects. Inhibition of PDE3 also induces dilation of the fetal and postnatal ductus arteriosus in humans. This effect is mediated by an increase in cAMP and cGMP in vascular smooth muscle  is expressed heavily in ductal SMC and acts as a calcium channel which increases intracellular Cain utero .in utero are summarized in Factors that maintain ductal patency Functional closure of the ductus occurs by 8 h in 44% of normal term infants and almost 100% are closed by 72 h. . The ratConstriction of the ductus is maintained via3 factors:4 receptors on the ductal wall.a) Decreased concentration of prostaglandins by the loss of placental source and increased removal by the lungs, and decreased number of EPb) Increased arterial oxygen pressure.c) Decreased pulmonary vascular resistance resulting in decreased blood pressure within the lumen of the DA.2 concentrations drop rapidly due to removal of placental production and increased metabolism in the lungs   . The act15-PGDH) . 15-keto to PGE2 . On the  to PGE2 . Bradykitriction .2 is higher than that in the term infant, which is attributed to increased binding to EP2, EP3, and EP4 receptors  and should be considered one of the body's major systems such as cardiovascular, nervous or endocrine systems .2  especially H2O2, which changes the cellular redox potential and alters the function of redox-sensitive genes, second messenger systems and oxygen-sensitive potassium channels, which control SMC membrane potential. Production of adenosine triphosphate (ATP) through oxidative phosphorylation increases with rising oxygen levels and inhibits KATP channels  oxidase. Proximal electron transport chain is the major oxygen sensor in the ductal SMC. This is reflected in the observation that electron transport chain inhibitors  mimic hypoxia and constrict the ductus, as well as activating the carotid body. When POchannels . These cchannels .2induced gene expression of ion channels  and T type (CaT) calcium channels, and opening of inositol triposphate (IP3) sensitive sarcoplasmic reticulum calcium stores which release calcium  expression, which promotes phosphorylation of myosin phosphatase inhibiting MLCP. Activation of the Rho-kinase pathway induces calcium sensitization, which sustains ductal constriction through positive feedback mechanism . Stimulation of these receptors induces IP3 production by phospholipase c, which leads to an increase in intracellular Ca2+ (Oxygen stimulates the release and synthesis of endothelin-1 (ET-1), acting on type A receptors . This is not profound enough to cause cell death but interferes with the ability of the ductus to close .Glutamate promotes ductal constriction by glutamate inotropic receptor subunit 1 (GluR1)-mediated noradrenalin production in animal models . It may +2 from endoplasmic reticulum, and inhibits the production and activity of prostaglandins. It is used frequently in extremely low birth weight infants for the prevention and treatment of apnea and bronchopulmonary dysplasia. However, it does not affect the contractile response of the ductus at therapeutic concentrations  plays an important role in regulating the body fluid volume and blood pressure. It is secreted from the ventricles in response to increased cardiac stress and increases intracellular cGMP, inducing vasodilation. In PDA, the volume of left-to-right shunting is associated with the BNP level and higher levels predict a hemodynamically significant PDA and a poor response to indomethacinin preterm infants , 64. BraProgesterone regulates prostaglandin synthesis and prostaglandin sensitivity of smooth muscle cells. Progesterone withdrawal in late pregnancy coincides with the increasing sensitivity of ductus arteriosus to constriction by indomethacin . This ef2 levels after birth, which impairs ductal vasodilation and a concomitant rise in oxygen which leads to vasoconstriction. Intracellular calcium increases, secondary to inhibition of Kv channels and KATP induced membrane depolarization. Increased calcium within the cell activates Ca2+–Calmodulin dependent MLCK activation, which phosphorylates MLC , notch signaling, Vascular Endothelial Growth Factor (VEGF) and fibronectin coupled with hyaluronan. The proliferation of SMCs is mediated by retinoic acid and notch signaling. Mounds are formed by expansion of the neointima by hyaluronan, which creates a suitable space for migrating SMC from the muscle into the intima and proliferating endothelial cells. The process begins with the accumulation of hyaluronan beneath the endothelial cells, accompanied by loss of laminin and type IV collagen and their separation from the internal elastic lamina. Influx of water into the hyaluronan widens the subendothelial space. Hyaluronan potentiates the migration of SMCs to the subendothelial space through hyaluronan binding protein, synthesized by SMCs  pathway in the ductal wall  has many pro-inflammatory effects, but also inhibits SMC proliferation and hyaluronan accumulation during the remodeling process of the ductus. The up-regulation of IL-15 in the ductus is mediated through the prostaglandin pathway. IL-15 upregulates the expression of EPal cells . In lateal cells .Notch receptors are critical for cell differentiation and there are 4 Notch receptors in humans. Notch 2 and Notch3 receptors are abundant in smooth muscle and they regulate proliferation, migration and angiogenesis in the vasculature. A decrease in the number of Notch receptors in SMCs is associated with downregulation of gene expressions related to contractile elements . Notch s2, IL-15, and oxygen , which stimulates hyaluronan production in the SMCs  is necessary for recruitment when physiologic shear forces are present and this interaction is only possible when the flow is very slow. Therefore, leukocytes can adhere to the ductal wall only after vasoconstriction and loss of ductal flow has occurred , 100 MonDuring the initial process of vasoconstriction, detachment of endothelial cells triggers the adhesion of platelets to the “injured” site, forming a platelet plug, which further blocks the lumen. Mice with disrupted platelet function or administered antibody against platelets have impaired ductus remodeling . The effAlthough they originate from the same neural crest lineage, there are major transcriptional differences between the DA and aorta. Several studies highlight the effect of genetic predisposition to the patency of DA. These include either the prostaglandin pathway , or vascular smooth muscle development or structure. Most of these information is obtained through mouse models. Overall, there are more than 100 genes are effective in the development of DA , 109.2 synthase to prostaglandin H2, which in turn is metabolized by PGE synthase to prostaglandin E2 (PGE2). Prostaglandin endoperoxide synthase 1 (Ptgs1 or COX1) and (Ptgs2 or COX2) is the rate limiting enzyme in the production of prostaglandins from arachidonic acid. Mice deficient in Ptgs1 have normal survival after birth whereas 35% of mice deficient in Ptgs2 die shortly after birth because of PDA (2 binds to the cell through its receptor EP4 (Ptger4) and mediates vascular smooth muscle relaxation and intimal thickening. Mice lacking these receptors die due to PDA (Hpgd gene encodes hydroxyprostaglandin dehydrogenase 15-(NAD) which is responsible for PGE2 metabolism. The deletion of this gene leads to elevated levels of PGE, leaving the DA open and resulting in death (Arachidonic acid is metabolized by PGHe of PDA . If bothe of PDA . Prostage to PDA . Furtherin death . Mice dein death .Pde5a mediates vasoconstriction in SMC of DA and its expression decreases in 6 h after birth (The effects of prostaglandins are mediated through cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). Phosphodiesterases degrade these compounds and several genes have been proposed for phosphodiesterases. er birth .The genes upregulated during ductal closure are phosphodiesterase 4B (PDE4B), desmin pyruvate dehydrogenase kinase isozyme 4 (PDK4), and insulin-like growth factor binding protein 3 (IGFBP3). Downregulated genes include cyclin-dependent kinase inhibitor 1C (CDKN1C) and alpha-2-glycoprotein1 (AZGP1). PDE4B limits the accumulation of cAMP, minimizing the vasodilator effect of endogenous prostaglandins. Therefore, increasing PDE4B would facilitate ductal constriction. Desmin is a component of the cytoskeleton and its increase indicates the maturation of SMCs. PDK4 and IGFBP3 regulate cell proliferation and apoptosis, which are operative in ductal closure. Increased transforming growth factor β-receptor II regulates PDK4 also , 116. WiMyh11) have delayed closure of DA  and its effector Rock2 is upregulated in the newborn DA and inhibition of this system may result in the patency of DA  and a subunit of L-type calcium channels (Cacna2d1) are highly expressed in the DA (Kcnk3 and Abcc9) are also upregulated in the DA (BKca) are abundant in vascular smooth muscle and they are expressed significantly higher in DA . There are 2 membrane receptors for ET-1, ET infants , 126.rs2234693, prostaglandin I2 synthase gene rs493694)( and another polymorphism in the interferon gamma gene rs2430561 is associated with a decreased risk of PDA and bronchopulmonary dysplasia (rs1056567) gene, angiotensin II type 1 receptor AGTR1 gene and TFAP2β (rs987237) gene, are associated with increased risk of PDA (Agtr2) is upregulated in late preterm infants also , and fibronectin (Fn1) genes which may draw attention in the future  and the pressure gradient between both ends , and inversely proportional to the blood viscosity and length of the tube . Moreover, increased venous return from the lungs to the left ventricle will cause an increase in the left ventricular end-diastolic pressure, which will increase atrial natriuretic peptide (ANP), with evolution of pulmonary venous hypertension and pulmonary hemorrhage. On the other hand, left to right shunting at the ductus will decrease the systemic perfusion  leading to systemic hypoxia and organ dysfunction. Shorter diastolic time and increased myocardial oxygen demand may result in endocardial ischemia .There is scarce information on the effects of dilation mediated by the mechanical force of the flow, or through shear forces and shear stress. Although there is no clear evidence, it may be speculated that there are some mechanosensors in the ductal wall, which can sense the biomechanical changes in the lumen and regulate ductal closure. The effect of laminar flow vs. turbulent flow across the ductus is also understudied. In case of laminar flow, endothelial cells are less likely to proliferate and nitric oxide synthase and prostacyclin are upregulated , 147. HoA sharp decrease in pulmonary artery pressure, as it happens after surfactant replacement therapy may increase the left-to-right shunt through the ductus, leading to pulmonary overflow and hemorrhage. In preterm infants, increased pulmonary fluid and protein load is eliminated by an increased lung lymph flow, named as “edema safety factor.” Therefore, ductal closure within the first 24 h of birth do not have any significant effect on the course of respiratory distress syndrome. However, if ductus remains open for several days, this compensation cannot be maintained and alteration in the pulmonary mechanics and pulmonary edema ensue. The impact of patent ductus arteriosus on patient outcomes seems to be a function of magnitude of shunt, rather than its mere presence. Closure of the ductus improves lung mechanics in these infants . Early d2. Phospholipase A2 stimulates the release of arachnidonic acid precursors needed for prostaglandin synthesis; thus in response to steroids, prostaglandin production is inhibited  or the peroxidase pathway (acetaminophen). The infant's gestational age, as well as the infant's race, ethnicity, growth, and exposure to antenatal corticosteroids modify the effects of prostaglandins and determine the rate of drug-induced closure , 169.2 receptors are down-regulated after birth. However, in the preterm infant, the production of receptors increases, rendering the ductus susceptible to vasodilation and resistant to indomethacin or ibuprofen. COX1 and COX2 expression increases with advancing maturity in the fetus  inhibits POX enzyme which catalyzes the conversion of PG2 to PGH2, without any peripheral vasoconstriction .Some authors have suggested to use a combination therapy of ibuprofen and paracetamol as a first line therapy for ductal closure. However, preliminary reports suggest that the effectiveness of combination therapy does not differ compared to monotherapy of either drug . In anotOther non-steroidal anti-inflammatory drugs such as aspirin, metamizol, nimesulid, and diclophenac have constrictor effects on the ductus –203.Glucocorticoids have also vasoconstrictive effects on the ductus, as well as synergistic effects with non-steroidal anti-infective drugs (NSAIDs), as a result of decreasing the sensitivity of the ductal SMCs to prostaglandins  Figure . In the There are many substances and foods with anti-inflammatory and anti-oxidant properties, such as those rich in polyphenols or flavonoids. For example, 30–40% of solid extract of green tea composes of polyphenols, mainly catechins. They exert their anti-inflammatory effects through the inhibition of endogenous prostaglandins. Similar effects have been reported with black tea, resveratrol, mate tea, orange juice and dark chocolate . ExcessiATP channel inhibitor, used for treating diabetes. It is shown that DA constricts in response to glibenclamide, and constriction is inhibited by diazoxide, a KATP channel activator (ATP channel genes, have PDA, often resistant to medical treatment, requiring surgical closure most of the time (ATP channels may modulate DA tone.Genetic therapies may include endothelin-signaling genes or blockage of potassium channels. Channel isoforms have distinct pharmacological and physiological characteristics, making them ideal targets for ductal-selective therapies . Glibencctivator . Childrethe time . Agonist4 inhibition (Novel treatment strategies for PDA include combined use of ibuprofen and paracetamol , EP4 inhhibition , NOS inhhibition , enhancihibition , glutamahibition  especial1 is the main drug used for vasodilatatory effects of the ductus. PGE1 binds to the EP4receptor which increases intracellular cAMP. Milrinon is a phosphodiesterase 3 inhibitor, which can also increase cAMP levels and dilate the ductus (PGEe ductus .Non-selective endothelin receptor antagonist TAK-044 and inhibition of Notch pathway may have vasodilating effects on the ductus , 91. Nat2 breakdown (Antenatal corticosteroids are used for the maturation of preterm lung, but their effects on the development of patent ductus arteriosus, intracranial hemorrhage, and necrotizing enterocolitis are also well-studied . Howeverreakdown .4 which is a calcium channel blocker may result in delayed ductal closure in infants (4 exposure is not associated with an increased risk of hemodynamically significant PDA; in fact, it may be associated with a decreased likelihood of hemodynamically significant PDA (ATP channel activator may induce ductal opening (2 production, thus dilating the ductus in animals but its effect is limited in humans (Antenatal MgSO infants . Howevercant PDA . Diazoxi opening . Furosemn humans .Ductus arteriosus is an intriguing and complex vessel with many distinct features. Understanding the precise mechanisms of regulation of the ductus is essential in order to improve individual pharmacological treatments. The debate about PDA should not be “treat all” or “treat none,” but rather, “who to treat.” This approach necessitates individualized treatment. Treatments targeting endogenous ductus regulation pathways other than prostaglandins, such as cell cycle regulators, transcription factors, and epigenomic regulators should be developed. Since vascular constriction and ductal wall remodeling are required for complete closure, treatments that favor vasoconstriction and remodeling would also be ideal for infants with persistent PDA. Novel therapies for ductal closure in preterm infants should also focus on a clear understanding of genes which are operative on ductal patency, as new genome-wide studies uncover genes associated with PDA. Evolving pediatric clinical pharmacology heralds next generation solutions for unresolved issues of today, such as drugs with selective DA effects, without any side effects.FO is responsible for the design, research, writing, and editing of the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Radiotherapy is one of the most common treatments for oral cancer. However, in the clinic, recurrence and metastasis of oral cancer occur after radiotherapy, and the underlying mechanism remains unclear. Cancer stem cells (CSCs), considered the “seeds” of cancer, have been confirmed to be in a quiescent state in most established tumours, with their innate radioresistance helping them survive more easily when exposed to radiation than differentiated cancer cells. There is increasing evidence that CSCs play an important role in recurrence and metastasis post-radiotherapy in many cancers. However, little is known about how oral CSCs cause tumour recurrence and metastasis post-radiotherapy. In this review article, we will first summarise methods for the identification of oral CSCs and then focus on the characteristics of a CSC subpopulation induced by radiation, hereafter referred to as “awakened” CSCs, to highlight their response to radiotherapy and potential role in tumour recurrence and metastasis post-radiotherapy as well as potential therapeutics targeting CSCs. In addition, we explore potential therapeutic strategies targeting these “awakened” CSCs to solve the serious clinical challenges of recurrence and metastasis in oral cancer after radiotherapy. Currently, the choice to treat oral cancer commonly includes surgery, radiotherapy and/or chemotherapy. Although strategies for treating oral cancer have continued to improve in recent decades, the prognosis of oral cancer remains low, with a long-term disease-free survival of ~50%.5 In oral cancer, radiotherapy is often selected for early-stage oral cancer patients as a single treatment or for advanced patients in combination with surgery.7 However, the main challenge in radiotherapy for oral cancer remains locoregional recurrence and/or distant metastasis, which is the major reason that cancer-associated mortality is still high worldwide.10 As reported, the median overall survival is only 6–9 months for head and neck cancer patients with relapse post-radiotherapy,12 and 10%–25% of head and neck cancer patients are diagnosed with distant metastasis after radiotherapy;10 this situation has a poor prognosis, with a median overall survival of 3–4 months and 1-year-free survival of <5%.13 Compared to hypopharyngeal and laryngeal cancers, oral cancers have a higher incidence of local relapse and shorter survival when distant metastasis occurs.14 Therefore, strategies to address relapse and metastasis of oral cancers post-radiotherapy need to be urgently developed.Radiotherapy is a critical therapy for cancer patients that causes DNA damage directly via ionising radiation (IR) or by indirectly generating oxidative damage via reactive oxygen species (ROS), thereby leading to cancer cell destruction.16 CSCs are considered to have innately higher radioresistance, invasive capacity and metastatic capacity than their differentiated cancer cell counterparts. Recently, increasing experimental and clinical studies have provided evidence in heterogeneous cancers that a very small number of CSCs surviving after treatment are able to repopulate a tumour at or near the primary cancer site and initiate metastasis development.19 Complicated cellular and molecular mecha-nisms, such as stemness maintenance, epithelial–mesenchymal transition (EMT) and reactive oxygen species (ROS) production, are involved in the process of CSC initiation and facilitation of cancer recurrence and metastasis after treatment.23 However, little is known about how CSCs play a role in oral cancer relapse and metastasis after radiation treatment. Herein, we will review the role of CSCs and how CSCs result in oral cancer relapse and metastasis after radiotherapy, as well as the potential mechanisms involved and relevant therapeutic targets.It is widely believed that cancer stem cells (CSCs), a small population of cancer cells with self-renewal capacity and multipotent differentiation potential, may be a main cause of cancer relapse and metastasis post-radiotherapy.27 CD44, a transmembrane glycoprotein, is one of the most common markers used for CSC enrichment28 and has been shown to enhance proliferation and survival by sequentially activating the MAPK and P13/AKT pathways.30 CD98+ subpopulations have been reported to express high levels of cell cycle and DNA repair genes and to have tumorigenicity in immunodeficient mice.24 CD133, a glycoprotein with 5 transmembrane domains, has been identified as a CSC marker in oral cancer; it appears to be involved in angiogenesis and has been shown to be negatively correlated with the prognosis of oral cancer.26 ALDH1, an enzyme responsible for detoxification of intracellular aldehydes, has been considered a specific marker of CSCs. ALDH1+ cells play a role in the development, metastasis and therapeutic resistance of head and neck squamous cell carcinoma (HNSCC).31 Generally, any stem cell-related marker can be used alone or in combination with other markers to sort oral cancer cells with stem cell characteri-stics. However, the specificity of CSC cell markers has been questioned.32 Taking CD44 as an example, it has been reported that CD44 is also highly expressed by the majority of normal oral epithelial cells and in oral mucosal tissue.34 Therefore, it is becoming more common for sorting of CSCs to be enhanced with the use of two or more enrichment markers simultaneously. Some studies indicate that the tumorigenicity of dual-marker-positive cells in vivo is higher than that of single-marker-positive cells, as is their resistance to cancer treatment.37 However, the lack of one stemness-related marker does not truly mean that cancer cells have no stem cell characteristics because CSCs are heterogeneous.40 To overcome the limitation of CSC markers in oral cancer, additional confirmation with a function-based enrichment method is a feasible strategy.Being able to sort and identify the CSC subpopulation is a prerequisite of further CSC research but is also a major challenge. Stem cell-related markers such as CD44, CD98, CD133 and ALDH1  are often used for CSC sorting and identification in oral cancer.41 Studies have indicated that CSCs that can form spheres retain their stem cell properties for several passages, while most differentiated cells die from anoikis.43 It has also been reported that sphere formation assays can be used to successfully enrich CSCs from primary oral cancer tissues or oral cancer cell lines.43 However, not all primary oral cancer cells can successfully form spheres,44 and the purity of CSCs obtained by this method has been questioned, leading to the suggested use of an additional enrichment step before the sphere-forming assay is performed.45The most widely used method of function-based CSC enrichment is the sphere formation assay, in which CSCs can grow non-attached in serum-free medium and form non-adherent 3-dimensional (3D) tumour sphere structures, a characteristic reminiscent of the ability of CSCs to initiate tumours and maintain tumour progression.44 and DNA dyes such as Hoechst 33342.45 In oral cancer, the CSC subpopulations called side populations (SPs) are identified based on efflux of Hoechst 33342 by flow cytometry or fluorescence-activated cell sorting (FACS), and SP cells have more clonal, invasive and tumorigenic ability than non-side population cells.47 The advantage of utilising SP sorting as a method to enhance the CSC subpopulation is that it does not require any specific stem cell markers and can be used to enrich CSCs from various tumour tissues and cancer cell lines. However, it is limited to some extent in that the sorted SPs are not homogenous. In addition, the application of this method is technically limited by low specificity, low purity of sorted cells, dye toxicity, variability in dye concentration and dyeing time.48Additionally, increased drug resistance, an important feature of CSCs, may serve as an alternative factor that can be utilised for CSC enrichment; this increased drug resistance of CSCs is achieved partly due to their high expression of ATP-binding cassette (ABC) transporters, which enable CSCs to efficiently dispel chemotherapeutic agentsGiven that the heterogeneity of oral CSCs is high and the specificity of markers is low, sorting and identifying oral CSCs is still challenging. Both enrichment methods based on stem cell markers and those based on functions have their shortcomings; thus, combining the existing enrichment methods to increase accuracy has been suggested Table .Table 1M16 Given this, the response of CSCs to ionizing radiation is critical to the prognosis of cancer patients post-radiotherapy.It is widely accepted in the CSC hypothesis that cancer grows as a hierarchy resembling normal tissue, with a small number of cancer stem cells functioning at the top of the hierarchy. Briefly, in this hierarchical CSC model, the ability to initiate tumorigenesis and generate heterogeneous cells in primary tumours is fully encompassed by the CSC population but absent in all differentiated progeny of CSCs Fig. .16 Given50 This is consistent with the prevailing view that malignant tumours contain dormant cells that are not sensitive to ionising radiation.51 It has been reported that even though a large number of differentiated tumour cells are killed by radiotherapy, the dormant cells considered to have some characteristics of CSCs can survive, and these cells are associated with subsequent tumour recurrence or metastasis.51 Interestingly, it is generally believed that in advanced cancer, most CSC populations are in a quiescent or dormant state.55 Studies have demonstrated that approximately one-third of CSCs in glioma and breast cancer cell lines are dormant but enter the cell cycle after radiation, whereas some non-tumorigenic cells (differentiated tumour cells) can become senescent after exposure to radiation.57 In other words, the quiescent CSC population can be “awakened” by ionising radiation to initiate proliferation and differentiation. Radiotherapy can not only cause dormant CSCs to enter the cell cycle but also induce them to develop a series of malignant phenotypes and carcinogenic metabolism.58 Thus, only if all CSCs are eliminated can tumours be permanently eradicated after radiation treatment.59Notably, active cell proliferation is a prerequisite for effective chemotherapy and radiotherapy of tumours, and any senescent and quiescent (not only CSCs) cells can be resistant to these therapeutic regimens.61 In addition, radiation can promote reversible transformations between stem and non-stem cells such that new CSCs can be generated from normal and neoplastic non-stem cells,66 resulting in an increase in the number of CSCs and the coexistence of different types of CSCs, leading to tumour heterogeneity.70 It has been reported in breast cancer that the absolute number of CSCs is elevated after exposure to ionising radiation, which is not able to be simply explained by the preferential killing of non-tumorigenic cells by ionising radiation.49 In addition, it was further confirmed by the same research group that radiation-induced upregulation of the embryonic transcription factors Sox2, Oct4, Klf4 and Nanog in polyploid cells in turn reprogrammes non-tumorigenic cancer cells to acquire CSC properties.68 Other scholars also observed that the expression of Sox2, Oct4 and Nanog was upregulated in lymphoma cells with p53 mutations after radiation.69 It has also been indicated in two hepatocellular carcinoma cell lines that radiation induces upregulation of Oct3/4 and Sox2, resulting in the acquisition of a CSC phenotype.67 Consistent with these results, radiation could induce the dedifferentiation of oral cancer cell lines, leading them to obtain a CSC phenotype.70 These findings suggest that differentiated cancer cells acquiring a CSC phenotype is a direct response to radiation rather than a random incidence. Therefore, we propose that in addition to “awakening” quiescent CSC populations, ionizing radiation can also “awaken” some cancer cells with potential stemness, reverting them to a stem-cell-like state.Several studies have shown that radiation treatment preferentially kills non-tumorigenic cells, thus enriching CSCs.In summary, it is a major barrier to successful radiotherapy that irradiation can “awaken” cancer stem cells. Current research outcomes suggest that ionising radiation cannot completely kill dormant CSCs because of radioresistance, but it does awaken them, causing them to enter the cell cycling, which further leads to malignant behaviours Fig. . In addi73 Whether alone or as part of combination treatment, radiotherapy still plays an important role in treating oral cancer at any stage of progression.7 However, some patients with oral cancer receiving radiotherapy who initially show obvious beneficial effects in terms of shrinkage or eradication of their primary tumours still rapidly develop local tumour recurrence, regional lymph node metastasis or distant lung metastasis.75 It has been reported that compared to other HNSCC tumours, such as hypopharyngeal and laryngeal cancers, oral cancers have a relatively higher recurrence rate postradiotherapy, especially advanced oral cancer, and once tumour metastasis occurs following radiotherapy, the prognosis of patients is extremely poor.14Radiotherapy remains one of the most common therapeutic approaches for the majority of cancer types. However, it has been reported in some studies that radiotherapy is also able to promote relapse and metastasis.76 Compared to differentiated cancer cells, undifferentiated CSCs innately have higher radiation resistance, which is possibly because ionising radiation preferentially kills proliferating tumour cells but is ineffective in quiescent CSCs, as they are in a dormant state. In fact, there is abundant evidence supporting that CSCs play a critical role in relapse and metastasis after radiation in several cancer types.19 Studies on glioma cells and breast cancer cells have found that radiation could only effectively kill non-CSCs, while CSCs survived due to their resistance to radiotherapy and were awakened from their dormant state, increasing rapidly after a period of time post-radiotherapy.57 It has also been found in breast cancer that the proportion of CSCs increases significantly after ionising radiation, and the CSCs show enhanced proliferation shortly after treatment, which further results in rapid tumour repopulation.18 All the findings mentioned here suggest that the surviving CSCs can maintain the ability to proliferate during intervals between radiotherapy applications or after radiotherapy, eventually inducing tumour relapse and resulting in an increased proportion of CSCs that enhance the malignancy of residual tumours. Consistently, findings in hepatocellular carcinoma indicate that compared to non-CSCs, irradiated CSCs are more capable of tumour formation when injected subcutaneously into nude mice.19 In addition, it has also been illustrated in non-small-cell lung cancer that cancer cells surviving radiotherapy obtain upregulation of markers of CSCs and mesenchymal cells, as well as the capacity to self-renew and to give rise to differentiated daughters,17 further confirming that the cancer cells surviving after radiation have a strongly malignant phenotype.Generally, the failure of radiotherapy for cancers is closely related to various factors, such as the radioresistance of cancer cells and the enhanced invasion and metastasis of tumours. Moreover, the radioresistance of CSCs is being increasingly recognised as a major factor leading to ineffective radiotherapy.77 It has been observed in studies on breast cancer, lymphoma and liver cancer that radiation can lead to upregulation of the expression of embryonic transcription factors by cancer cells, thus inducing the dedifferentiation of cancer cells into CSCs,69 which is potentially associated with relapse and metastasis post-radiotherapy. Additionally, cells with an IR-induced CSC phenotype have been confirmed to have stronger sphere-forming and tumour-forming abilities in mice,68 which means that the number of tumorigenic cells increases after radiation, potentially leading to more rapid tumour recurrence. In addition, it has been confirmed by evidence that CSCs have mesenchymal phenotypes, suggesting their high capacity for migration and invasion.80 IR can induce non-CSCs to have stronger migration activity, which is closely related to the acquisition of the CSC phenotype and radiation-induced epithelial–mesenchymal transformation (EMT),58 which is associated with relapse and metastasis.Although ionizing radiation preferentially kills proliferating tumour cells, nearly half of cancer cells can still survive after radiotherapy.83 Moreover, IR was found to play a significant role in the dedifferentiation of multiple oral cancer cell lines, and 5 days after radiation application, the proportion of CSCs in oral cancer cell lines increased significantly at different IR doses, as assessed by flow cytometry analysis. In addition, the sphere formation assay in this study showed that a single dose of 8 Gy significantly enhanced the sphere-forming ability of oral cancer cell lines.70 To draw a preliminary conclusion from the current evidence mentioned above, we believe that the response of oral CSCs to radiotherapy will be found to be similar to that of other CSC types, namely, that oral CSCs can be “awakened” by radiation, further resulting in higher proliferation, migration, invasion and radioresistance, which play a critical role in tumour relapse and metastasis post-radiotherapy.For oral cancer, it has recently been revealed that IR can promote the migration of several oral cancer cell lines, including Cal-27, SCC25 and FaDu.As mentioned above, CSCs are closely related to recurrence and metastasis after radiotherapy for oral cancer, especially the populations of radiation-activated CSCs and those cells that have undergone radiation-induced dedifferentiation to obtain a CSCs phenotype . However, little is known about how “awakened” CSCs result in oral cancer recurrence and metastasis after radiotherapy. Both cancer cells and their surrounding tumour microenvironment play a crucial role in cancer progression, and CSCs initiate tumorigenesis and enhance tumour development. Therefore, radiation-induced changes in CSCs themselves and in their environment should have an effect on recurrence and metastasis post-radiotherapy in oral cancer. Next, we will discuss the potential mechanism by which CSCs cause recurrence and metastasis of oral cancer after radiotherapy. The complex mechanisms are briefly summarised in Fig. 84 CSCs are considered to have greater radiation resistance than cancer cells, enabling them to survive radiotherapy in various ways, such as controlling the cell cycle, effective DNA repair and modified division patterns.85 The radioresistance of CSCs due to their innate stemness enables them to survive after radiotherapy, eventually leading to tumour recurrence or metastasis.The inherent function of stem cells, permanently providing genetically correct cells to replenish functional tissues, suggests that stem cells have a natural advantage in resisting DNA damage; this resistance aids them in response to radiotherapy, and the radioresistance of these cells is a direct reflection of their inherent ability of DNA repair.55 Studies in glioma and breast cancer have found that one-third of CSCs are dormant and do not enter cell cycling until exposed to ionizing radiation,50 suggesting a mechanism by which “awakened” CSCs could cause tumour recurrence after radiation. It is also consistent with the idea we discussed above that ionizing radiation is more effective in eradicating rapidly dividing and proliferating cancer cells than dormant or quiescent cells, which helps such dormant cells survive after radiation. Moreover, in both preclinical in vivo models and cancer patients, dormant cells have been confirmed to survive existing therapies, including radiotherapy,50 which may be the cause of tumour recurrence. In addition to radioresistance due to dormancy, irradiated CSCs also show a stronger ability for DNA repair than differentiated cancer cells. With the detection of gH2AX, a specific DNA damage marker,86 it has been reported that in breast cancer, there was no significant difference in the number of gH2AX foci between CSCs and other tumour cells when detected immediately after exposure to ionizing radiation; however, after 48 h, the number of gH2AX foci in CSCs was significantly lower than that in other cancer cells,87 indicating that CSCs had a higher capacity for DNA repair. In general, having innate resistance to radiotherapy and high DNA damage repair can synergistically enhance CSC survival during radiotherapy.Generally, several studies have reported that quiescent CSCs proliferate more slowly than non-CSCs.65 Generally, CSCs undergo an asymmetrical division, giving rise to a CSC capable of infinite proliferative potential and a differentiated daughter cell with limited division.88 However, when tumours are damaged after treatment, such as chemotherapy and radiotherapy, symmetrical CSC division, in which both daughter cells are CSCs that can divide indefinitely, may predominate,89 accelerating tumour recurrence. As reported in breast cancer, the number of CD24−/CD44+ cells increased within a short period of time after radiation accompanied by Notch upregulation.18 Findings in glioblastoma showed that CSC enrichment was not only due to the radiation resistance of CSCs but also closely related to the increase in radiation-induced symmetrical division of CSCs, with a potential mechanism that may be related to the activation of the Sonic Hedgehog (SHH), Notch, Wnt and epidermal growth factor receptor (EGFR) signalling pathways.90 Radiation-induced Notch and EGFR pathway activation has already been confirmed to enhance self-renewal by increasing symmetric division.54Self-renewal, a type of cell division specific to stem cells that includes symmetrical and asymmetrical renewal, enables cells to divide indefinitely and retain their differentiation potential.91 and promoting EMT of CSCs while still maintaining their stemness.93 Therefore, the alteration of CSC division post-radiotherapy together with their innate strong self-renewal ability can guarantee the rapid proliferation of surviving CSCs after radiotherapy, further increasing the possibility of tumour relapse.In addition, the high expression of telomerase in CSCs helped maintain their self-renewal ability by playing a role in preventing DNA telomere shortening during cell division, thus allowing cells to proliferate indefinitely,95 has been confirmed to play an important role in cancer progression and response to cancer treatment.55 Briefly, the process of EMT endows epithelial cells with a mesenchymal phenotype that is characterised by the loss of epithelial morphology and markers  and the acquisition of mesenchymal markers . Therefore, it is widely believed that cancer cells that have undergone EMT have stronger migration activity than those that retain an epithelial phenotype.96 There is increasing evidence that CSCs have mesenchymal cell phenotypes,80 which can ultimately lead to tumour metastasis. It has been found in nasopharyngeal carcinoma that nasopharyngeal carcinoma cell lines surviving radiation treatment show decreased E-cadherin and increased vimentin, resulting in EMT occurrence with strong migration activity.6 It has also been demonstrated in other head and neck squamous cancer cell lines that X-ray radiation can promote EMT and enhance migration and invasion.97Epithelial–mesenchymal transition (EMT), a developmental process involved in embryogenesis, wound healing and organ fibrosis,101 TGF-β1 can be specifically induced by ionising radiation in cancers.103 Our previous study indicated that radiation could induce increased activation of the TGF-β signalling pathway as well as elevated ROS expression, facilitating oral cancer progression.104 It has also been verified through an in vitro study in oral squamous cell carcinoma that TGF-β1 can enhance the CSC phenotype of cancer cells.105 Findings in a cervical cancer study showed that TGF-β1 promoted EMT in cancer cells and helped them to obtain a CSC phenotype.80 Similarly, in hepatocellular carcinoma, TGF-β1 could achieve the same effect as in cervical cancer by downregulating TP53INP1 through miR-155.78 These studies suggest that the radiation-induced TGF-β signalling pathway plays an important role in EMT and obtaining a CSC phenotype, thereby promoting tumour metastasis.In addition, radiation-induced EMT of cancer cells has been confirmed in several studies and has been found to be mediated by complex molecular signalling factors, such as TGF-β and ROS.106 Moreover, it has been reported in pancreatic cancer that N-acetylcysteine (NAC), an antagonist of ROS, can reduce the stemness and EMT phenotype of drug-resistant pancreatic cancer cells.107 In addition, inflammatory cytokines, hypoxia-inducing factors and matrix metalloproteinases are all involved in ROS-mediated EMT cascades.110 These results confirm the key role of ROS in EMT, which indicates that the high concentrations of ROS stimulated by irradiation may promote EMT.ROS are also involved in TGF-β1-induced EMT in head and neck cancers. In oesophageal adenocarcinoma, TGF-β1 reduces the level of ferritin heavy chain (FHC), which in turn increases the levels of intracellular ROS and activates the p38 MAPK signalling pathway, further inducing EMT. Subsequently, it was further demonstrated that ROS deficiency was able to inhibit the migration of tumour cells.112 With increased CSC studies, the “niche” concept has been extended to CSCs, in which CSCs can be supported and induced by the connective tissue matrix and vascular tissue comprising CSC niches.114 In malignant tumours, CSCs niches promote the divisional dynamics of CSCs, enabling them to produce progenitor cells that further facilitate self-renewal of CSCs and maintain their initial development conditions.89 In addition, other cells in CSC niches can function in helping CSCs to metastasise, escape apoptosis, and alter cell division dynamics via activation of various signalling pathways, resulting in cell repopulation.117 It has been reported that nitric oxide produced by endothelial cells in the CSC niche can activate the Notch pathway in glioma, thus promoting self-renewal of CSCs and enhancing their tumorigenic ability in vivo.118 In hepatocellular carcinoma, endothelial cells exposed to radiation express elevated IL-4 to activate the ERK and AKT pathways, which promotes the migration and invasion of cancer cells as well as increases the size of the CSC population.119 Myofibroblasts in the CSC niche have also been found to activate the Wnt signalling pathway by expressing hepatocyte growth factor, thereby inducing cancer cell dedifferentiation into CSCs and expression of stemness-related genes.27It has already been confirmed that the anatomically distinct microenvironment containing normal stem cells, called the niche, is composed of a variety of different cells and the extracellular matrix (ECM), which protect normal stem cells and regulates their functions.120 Moreover, the vascular endothelium in oral cancer has also been demonstrated to play an important role in tumour progression; for example, interleukin-6 (IL-6) and epidermal growth factor (EGF) secreted by vascular endothelial cells could promote the stemness of cancer cells in the perivascular niche and improve their vitality, enabling them to escape apoptosis.117In HNSCC, the majority of CSCs are located perivascularly within a radius of 100 μm, suggesting that a perivascular niche of CSCs exists. In a tumour angiogenesis SCID mouse model, it was observed that specific ablation of tumour-associated endothelial cells resulted in a reduction in head and neck CSCs.121 both of which contribute to CSC radioresistance. While decreased angiogenesis is induced by irradiation in the CSC niche, increased hypoxia associated with growth factor and inflammatory factor expression also occurs, which may accelerate tumour recurrence and the generation of invasive CSCs that contribute to tumour metastasis.116 In addition, it is widely believed that most cancers have multiple clonal cell types related to CSC subpopulations in the same tumour, leading to tumour heterogeneity. Although it is not yet known whether these heterogeneous CSCs are generated by different niches, some studies suggested that more complex niches could promote the radioresistance of CSCs.122Similar to the perivascular niche, the CSC niche in response to radiation also provides a hypoxic microenvironment, to further keep CSCs in a quiescent state and to resist radiation-induced oxidative stress,The tumour microenvironment (TME) refers to the internal environment in which tumour cells are generated and survive, composed of tumour cells themselves; surrounding stromal cells, i.e. fibroblasts and immune and inflammatory cells; intercellular substances; microvessels; and complex molecules in the surrounding area. The TME has been confirmed to play a critical role in mediating the radiation resistance and migration of cancer cells. Ionising radiation can cause a variety of changes in the TME, including increased secretion of various cytokines by stromal cells and recruitment of immune cells, contributing to the self-renewal and stemness maintenance of CSCs as well as associated cancer progression.123 It is widely considered in the majority of cancers, including oral cancer, that CAFs could play a positive role in cancer progression through their interaction with cancer cells or paracrine signalling affecting cancer cells, including CSCs.127 In the study of lung cancer, it was observed that CAFs could activate IGF1R signalling in cancer cells, which further induced the expression of Nanog in cancer cells, contributing to the promotion of the stemness of CSCs, suggesting that CAFs in the CSC niche support cancer stemness.128 In addition, many studies have also focused on the effect of radiation on CAF functions and found that radiation can promote the secretion of various cytokines by CAFs with a positive role in cancer progression.132 It has been observed in colorectal cancer that radiation-activated CAFs promote the metabolic conversion of glutamine through IGF1R activation, which further aggravates cancer progression.130 In vitro findings in hepatic carcinomas showed that radiation could induce CAFs to secrete various cytokines, including tumour necrosis factor-α (TNF-α), IL-6, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), MMP2 and MMP9,131 promoting the survival, metastasis and self-renewal of CSCs. It has also been indicated in breast cancer in vivo that pre-irradiation of mice bearing breast cancer increased the number of circulating tumour cells  and the incidence of lung metastasis via the elevated expression of cyclo-oxygenase-2 (COX2) and IL-6 in the TME.133 Similarly, in squamous cell carcinomas, it has been shown that CAF-secreted TGF-β induced by irradiation can promote the migration and invasion of cancer cells in vitro71 and increase the number of CTCs and lung metastasis in breast tumours in situ.134Carcinoma-associated fibroblasts (CAFs), as a key component in the TME different from normal fibroblasts, have a modified phenotype via interactions with tumour cells.137 It was found in primary human glioma that the distribution of TAMs in the leading edge of invasive tumours was associated with the presence of CD133+ glioma CSCs and that TAMs significantly enhanced the invasiveness of glioma stem cells through paracrine TGF-β1.138 In addition, a large number of TAMs were found around CD44+ALDH+ cells in colon cancer and CD133+ALDH+ cells in lung cancer, which synergistically activated Sonic Hedgehog pathways in CSCs with IL-6 secretion.139Additionally, radiation can affect the recruitment of immune cells as well as their biological behaviour. After irradiation, tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) can rapidly infiltrate the irradiated site with increased secretion of cytokines and growth factors related to the stemness of cancer cells, thereby altering the growth and migration of cancer cells.117 In addition, TGF-β has also been confirmed to induce oral squamous cell carcinoma cells to take on a CSC phenotype by abolishing FOXO3a.105 Although there is an unfortunate lack of studies on the response of oral CSCs to radiotherapy to date, the results in the other cancers mentioned above suggest that the increased secretion of cytokines in the oral cancer TME following radiotherapy may contribute to the recurrence and metastasis of oral cancer post-radiotherapy by enhancing CSC properties.Similar findings in oral cancer patients showed that the level of serum IL-6 in recurrent patients was higher than that in patients with only primary tumours, with further preclinical study showing that the level of IL-6 was crucial for maintaining the self-renewal and tumorigenicity of CSCs in oral cancer.140 It has also been confirmed that hypoxia can enhance the migration of cancer cells,141 as well as maintain CSC stemness and promote EMT.142Intratumoural hypoxia is an important indicator of poor prognosis in cancer patients and can lead to severe progression, frequent metastasis and resistance to radiotherapy.144 Importantly, HIF-1 has also been confirmed to be an important mediator in hypoxia, maintaining the stem cell phenotype of CSCs.145 It has been reported that under hypoxic conditions, CSCs sorted from FaDu cells, an oropharyngeal cancer cell line, express HIF-1α faster than non-CSCs after radiation. In addition, the radiosensitivity of both CSCs and non-CSCs increased significantly after inhibiting HIF-1α expression even in a hypoxic environment.146It has been observed that radiation can induce significantly elevated levels of tumour hypoxia inducible factor-1 (HIF-1), which further increases the proportion of hypoxic cells in tumours to regulate tumour radiosensitivity.3 ROS can cause structural damage when they occur within a 2-nanometre range of cellular DNA.89 Notably, the ability of ROS to result in DNA damage is attributed to the oxidative stress of irradiated cells; therefore, hypoxic cells have stronger radiation resistance than cells in a normoxic environment.147 Therefore, IR-induced additional ROS are less lethal to hypoxic cells and may unexpectedly promote their malignant phenotype. For CSC populations in cancer, it has been found that CSCs can prevent oxidative damage to DNA from ROS by increasing the production of free radical scavengers, further bolstering their inherent radiation resistance.148 In addition, CSCs in tumours are more localised to hypoxic areas than to those with normal oxygen concentrations, which will further weaken the lethality of ROS against CSCs and enhance CSC radioresistance.146 In summary, radiation-induced ROS production and hypoxic conditions have a close relationship in response to radiotherapy by affecting CSC radioresistance.It is well known that the accumulation of ROS can result in DNA damage in the majority of tumour cells in response to radiotherapy.As reviewed above, relapse and metastasis of oral cancer after radiotherapy remain major challenges. Given the close relationship between CSCs and relapse and metastasis, particularly the “awakened” CSC subpopulations, it is necessary to develop therapeutic strategies targeting CSCs to improve oral cancer prognosis. Although there is limited knowledge on therapeutic strategies targeting oral CSCs, we will briefly discuss current strategies, including treatments targeting CSC markers, CSC self-renewal pathways, CSC-associated EMT and the CSC niche.149 In preclinical trials in HNSCC patients, it has been shown that combined application of both an anti-CD44v6 monoclonal antibody and maytansinoid (DM1) in patients together with segmentation radiation could significantly improve the control of permanent local tumours.150 Additionally, in a previous clinical study of patients with refractory squamous cell carcinoma of the head and neck or oesophagus, it was observed that combined treatment with bivatuzumab, a human monoclonal antibody against CD44v6 and mertansine (DM1), led to better efficacy than monotherapy with either agent, although this clinical study was discontinued after a subject died of toxic epidermal necrolysis.151 It is worth noting that the identification of CSC markers should take into account the specificity of the markers. Given that there is a large amount of CD44 in normal oral tissues,34 it is difficult to use anti-CD44 antibodies targeting CSCs in oral cancer. Additionally, it was found that using lentivirus transfection to silence CD133 in oral squamous cell carcinoma could help decrease the therapy resistance of oral cancer significantly.26 Although no clinical studies have been conducted on targeting CSC markers in combination with radiotherapy in oral cancer, the studies mentioned above have provided us with good insight.CSC-targeted therapy can be achieved by directly targeting overexpressed CSC-related molecules. It has been reported that pancreatic cancer patients with high CD44 expression had significantly shorter overall survival than those with low CD44 expression. An anti-CD44 antibody could inhibit the growth and metastasis of transplanted pancreatic tumours in mice, and the anti-CD44 antibody combined with radiotherapy could reduce tumour relapse after radiotherapy.152 In addition, BMI1 has been found to be highly expressed by CD133+ glioblastoma cells, which promote DNA repair by preferentially activating the DNA double-strand break (DSB) response mechanism; in addition, BMI1 deletion can seriously inhibit the DSB response, resulting in increased sensitivity to radiation, suggesting that pharmacological inhibition of BMI1 combined with radiotherapy may provide an effective means of targeting CSCs.153 A similar finding of BMI1 targeting CSCs was observed in CD44+ nasopharyngeal carcinoma: when overexpressed BMI1 was knocked out in CD44+ cells, DNA damage repair was inhibited, and cell apoptosis increased after radiotherapy.154 In addition, in colorectal cancer, downregulation of BMI1 inhibited the self-renewal ability of CSCs, leading to the loss of their tumorigenic potential, while the use of a small molecular inhibitor of BMI1 could cause long-term damage to colorectal xenograft tumours in mice.155 Moreover, BMI1 is also highly expressed in CSCs of oral squamous cell carcinoma. BMI1 was able to suppress the ability of oral CSCs to form cell spheres in vitro, and a BMI1 inhibitor could arrest the progression of xenograft tumours in association with a reduced proportion of CSCs.156 All these results suggest that strategies targeting CSC self-renewal, such as BMI1 inhibition, have great potential to be combined with radiotherapy in oral cancer for better prognosis.Their innate self-renewal ability is one of the most important mechanisms of CSCs causing tumour relapse after radiotherapy. Several developmental signalling pathways, such as SHH, Notch and Oct4, are active regulators of autophagy and proliferation of stem cells, providing potential CSC targets to treat cancers. By targeting SHH signalling, the autophagy regulator BMI1 has been proven to play a role in the self-renewal of stem cells.157 Thus, strategies targeting the EMT-related transformation of cancer cells into CSC-like cells in combination with radiotherapy should offer strong alternatives for improving prognosis in oral cancer. It has been revealed that miR-495 can weaken EMT phenotype acquisition in nasopharyngeal carcinoma cells and enhance their radiosensitivity by downregulating the expression of the cell surface protein GRP78.158 Signal transducer and activator of transcription 3 (STAT3), an important transcription factor induced by EMT, has been found to play a role in increasing IR-induced CSCs in pancreatic cancer. In addition, inhibiting the DNA binding activity of STAT3 could reverse its role in the increase in IR-induced CSCs and in turn promote the radiosensitivity of IR-resistant pancreatic cancer cells.159 Notably, metformin has been shown to inhibit EMT in a variety of tumours,162 correspon-dingly contributing to improving the efficiency of radiotherapy in mice xenografted with prostate cancer cells and colorectal cancer cells,163 as well as the radiosensitivity of breast cancer and prostate cancer patients, as observed in retrospective studies.165 In HNSCC, it has been observed that metformin can inhibit cancer progression by acting directly on CSCs through downregulation of cancer stemness signature-associated genes.166 It has also been found that metformin can prevent the occurrence of chemical-induced oral cancer and downregulate markers of oral cancer CSCs in mice.167 Therefore, targeting the CSC-associated EMT induced by radiation in oral cancer, such as with the use of metformin, is a promising strategy for radiosensitization.Radiation-induced EMT may be a key step in the acquisition of the CSC phenotype.169 In addition, elimination of tumour blood vessels results in increased sensitivity of CSCs to cytotoxic drugs.170 Additionally, the specific ablation of tumour-related endothelial cells induced by caspase-9 leads to a reduction in CSCs in head and neck cancers,120 suggesting that targeted elimination of the CSC perivascular niche combined with radiotherapy may be a promising oral cancer treatment. In fact, X-ray irradiation of lung adenocarcinoma caused a significant increase in vessel density during tumour formation and increased induction of c-kit phosphorylation in endothelial cells.171 Thus, the application of anti-angiogenesis drugs targeting CSC niches in combination with radiotherapy may be a promising therapeutic strategy in cancer, but further studies are needed.The CSC niche, a complex biological environment that CSCs depend on, has been confirmed to play an important role in the CSC response to radiotherapy. Targeting CSC niches is a potential alternative strategy to damage CSCs during radiotherapy. As mentioned previously, most CSCs in HNSCCs, including oral cancer, are located in the perivascular region, suggesting that the targeted elimination of the CSC perivascular niche may be able to affect the prognosis of oral cancers treated by radiotherapy by reducing nutrient support and the secretion of signalling molecules required by CSCs. Some studies have shown that treating mice bearing cancer with bevacizumab, an anti-angiogenic drug, can result in failure of tumour angiogenesis and significantly reduce CSC numbers as well as tumour growth.As stated above, there is very limited knowledge to date on targeted therapy for oral CSCs, especially in combination with radiotherapy. However, there is no doubt that new approaches targeting CSCs in combination with radiotherapy may achieve better efficacy to treat oral cancer. Theoretically, it is feasible to directly target CSCs themselves or CSCs niches to achieve radiotherapy sensitisation Fig. . HoweverAs they are the “seeds” of cancer, the way CSCs respond to treatment is crucial to the prognosis of tumours. Given the results focusing on CSCs and radiation treatment in oral cancer and other cancer types mentioned above, we can preliminarily draw the conclusion that oral CSCs can survive radiotherapy significantly better than non-stem cells, and surviving, quiescent CSCs can be “awakened” by ionising radiation, upregulating cell proliferation, which plays a key role in tumour relapse and metastasis. In addition, oral cancer cells with high malignant potential  may also be “awakened” by radiotherapy to dedifferentiate into CSCs or to obtain a CSC phenotype with stemness-related marker expression. As reviewed, the “awakened” CSCs induced by radiation may contribute to oral cancer recurrence and metastasis post-radiotherapy due to their inherent radiation tolerance, DNA repair ability, self-renewal properties and innate mesenchymal phenotype, which endows them with the potential for invasion, and these properties can be reinforced by the CSC niche and tumour microenvironment. However, there is no doubt that our recognition of the role of oral CSCs in tumour recurrence and metastasis post-radiotherapy is still insufficient. More research on how CSCs are “awakened” by radiation and the mechanisms involved in oral cancer relapse and metastasis should be carried out to help explore more potential strategies for targeting oral CSCs, which will ultimately contribute to a better prognosis of oral cancer after radiotherapy.


Respiratory syncytial virus (RSV) is often the first clinically relevant pathogen encountered in life, with nearly all children infected by two years of age. Many studies have also linked early-life severe respiratory viral infection with more pathogenic immune responses later in life that lead to pulmonary diseases like childhood asthma. This phenomenon is thought to occur through long-term immune system alterations following early-life respiratory viral infection and may include local responses such as unresolved inflammation and/or direct structural or developmental modifications within the lung. Furthermore, systemic responses that could impact the bone marrow progenitors may be a significant cause of long-term alterations, through inflammatory mediators and shifts in metabolic profiles. Among these alterations may be changes in transcriptional and epigenetic programs that drive persistent modifications throughout life, leaving the immune system poised toward pathogenic responses upon secondary insult. This review will focus on early-life severe RSV infection and long-term alterations. Understanding these mechanisms will not only lead to better treatment options to limit initial RSV infection severity but also protect against the development of childhood asthma linked to severe respiratory viral infections. Studies have shown that up to 68% of infants may be infected with respiratory syncytial virus (RSV) in their first year of life, and nearly all children will be infected by two years of age . RSV infThere is currently no vaccine available or a sufficient option for antiviral therapy, partially due to our incomplete understanding of how severe immunopathology is developed. In the 1960s, a vaccine trial was conducted with suboptimal results; vaccinated infants became severely exacerbated upon exposure to live virus leading to severe disease as well as two infant deaths ,8,9. TheAlong with the initial disease complications associated with primary RSV infection, RSV-driven Th2-immunity skewing has also been implicated in leading to airway restructuring linked to exacerbated allergic responses later in life in animal models . Up to 4In this review, we will summarize several clinical and experimental studies related to severe RSV disease and subsequent risk for asthma and allergic responses later in life. The use of different experimental approaches in human and animal models have been crucial to understanding the role of RSV on chronic respiratory diseases. It is important to note that detailed mechanistic studies, especially those related to neonatal RSV infection, need to be conducted using animal models and/or in vitro cell systems due to necessary limitations on clinical testing and sample collection in human subjects. We will discuss how early-life RSV infection alters the immune response and may have long-term or systemic effects that can lead to severe complications throughout life. RSV is one of the most frequent viral causes of lower respiratory tract infection in children and is implicated in up to 80% of all bronchiolitis cases ,21. BronRSV predominantly infects ciliated epithelial cells in the airways but can also lead to infection in the subepithelium and neighboring immune cells, such as macrophages and dendritic cells ,26. The Initial RSV response is driven by innate cytokines, such as IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) that are released from the airway epithelial cells following infection ,33,34,35A nonpathogenic response requires a balance of antiviral and regulatory immune cells, and inappropriate immune responses have been linked to the development of pathologic responses following RSV infection in mice as well as using in vitro modeling in human cell lines ,29,42. FA significant producer of Th2 cytokines are the innate lymphoid 2 cells (ILC2), known to be involved in lung disease pathogenesis. ILC2 are a relatively rare population of innate cells of lymphoid origin, but unlike T cells or B cells do not contain an antigen receptor, and are therefore nonspecific innate responding cells . It has CCL5 was associated with bronchiolitis caused by respiratory syncytial virus (RSV) and RSV-subtype-A while the SNP rs1060826*NOS2 was associated with bronchiolitis caused by rhinovirus [TLR4, rs1898830*TLR2, rs7656411*TLR2, rs352162*TLR9, and rs187084*TLR9 as well as rs2280788*CCL5 were associated with severity of bronchiolitis [TLR4 (Asp299Gly and Thr399Ile mutations) represent risk factors for RSV infection [TLR4 (rs4986790 and rs4986791) [Other factors that can affect RSV immunopathology and the severity of the disease are genetic polymorphisms ,60,61,62inovirus , indicathiolitis ,59. TLR4In addition to severe disease following initial RSV infection, many studies have indicated that the immune system is persistently altered following this early-life infection, which may impact future immune responses later in life ,56,63,64Studies with murine neonatal RSV infection have demonstrated that there are persistent changes in the lung that include mucus production and increased immune cell populations that persist in the lung, including ILC2 ,48,49,56The detrimental role of increased ILC2 following RSV infection may be two-fold—1) as an inducer of pathogenic inflammation as described above and 2) in altering the structural/developmental process of the lung in neonates. The early-life lung in both humans and mice is predisposed to a type 2 immune environment for proper lung development to occur. This predisposition may enhance the detrimental effects of RSV by hijacking these programs, leading to severe immunopathology as well as improper lung development. IL-33-specific ILC2 have a known role for normal lung development ,69 and iThese studies highlight the role that the innate immune system has on viral immunity and possible long-term consequences on lung function and environment. These include alterations in not only innate immune cells themselves, such as myeloid cells  and ILC2 but also the cytokine milieu that they express, including increased expression of IL-33 and TSLP as well as IL-13 with concomitant inhibition of type-1 IFN. Thus, innate immune responses during early-life RSV infection appear to be central to the development of long-term lung environment as well as structural alterations within the lung . Trained immunity involves innate cells such as myeloid cells (including dendritic cells), natural killer (NK) cells, and innate lymphoid cells (ILCs) and their interactions with pattern recognition receptors (PRRs) and effector cytokines. In contrast to classical adaptive memory, the increased responsiveness to secondary stimuli during trained immunity is not specific for a particular pathogen and it is mediated through signals involving transcription factors, epigenetic reprogramming and metabolic alterations ,80,81. AMany studies have shown that myeloid cells are significantly altered following viral and bacterial infections ,86,87,88It has been shown that the numbers of DCs increase in the lung following RSV infection in both humans and mice ,95,96,97On the other hand, cDCs have been implicated in promoting the Th2 response by upregulation of CCR6 and CCL20 secretion leading to the recruitment of Th2 cells to the lung ,103. IntMany mouse and human studies have also identified expression of several DC-linked chemokines  that contribute to the pathogenesis of asthma, RSV-induced exacerbation, and the clearance and resolution of the ongoing pathology ,103,105.In addition, chemokine receptors  have been identified that regulate the recruitment of specific immune cells involved in the response to RSV and/or allergen-induced asthma-like responses ,106,107.Streptococcus pneumoniae while RV infection was associated with increased levels of essential and nonessential N-acetyl amino acids and high relative abundance of Haemophilus influenzae [The concept that our immune responses are influenced by environmental factors including microorganisms, pollution, diet and pathogen exposure that lead to shifts in microbiome and metabolic profiles, is important for understanding disease progression. Interestingly, a study of infants hospitalized due to bronchiolitis caused by RSV or rhinovirus (RV) infection determined that the microbiome and metabolic programming varied significantly between infants hospitalized due to RSV vs. RV . RSV wasfluenzae . These sfluenzae , therefo2O2 in the airways and was protective against RSV-induced pathology [Untargeted metabolic analysis of human airway epithelial cells during RSV infection has shown that several metabolites are altered after infection, with upregulation of nucleotides, amino acids, amino and nucleotide sugars, and metabolites of the central carbon pathway while the metabolism of lipids were downregulated . The autathology , suggestLactobacillus johnsonni (LJ) prior to RSV infection led to significant alterations of the bone marrow-derived dendritic cells (BMDCs) upon ex vivo analysis [Metabolic reprogramming of macrophages and dendritic cells following pathogen exposure have been identified in the literature ,116,117.analysis . These canalysis . Additioanalysis . TogetheEpigenetic modification of immune cells will be critical to understand both innate and acquired immune responses. Mouse studies have shown that the regulation of DC function can be modified by epigenetic changes to chromatin structure during maturation with CCR7  as well These data presented here along with studies describing the trained immunity phenomenon suggest that RSV infection is capable of altering the immune system in such a way that may lead to trained immunity in systemic populations within the bone marrow. These modifications are found at both the metabolic level as well as through epigenetic/transcriptional alterations  which toRSV continues to be a significant healthcare burden ,4,5,6 asThe studies described have elucidated novel and exciting aspects of RSV-driven immunopathology and suggest that it is imperative to consider regulating the innate immune system during RSV disease. Importantly, these studies have uncovered many pathways that may potentially be targeted and ultimately lead to better treatment options . Further


The root cap, a small tissue at the tip of the root, protects the root from environmental stress and functions in gravity perception. To perform its functions, the position and size of the root cap remains stable throughout root growth. This occurs due to constant root cap cell turnover, in which the last layer of the root cap is released, and new root cap cells are produced. Cells in the last root cap layer are known as border cells or border-like cells, and have important functions in root protection against bacterial and fungal pathogens. Despite the importance of root cap cell release to root health and plant growth, the mechanisms regulating this phenomenon are not well understood. Recent work identified several factors including transcription factors, auxin, and small peptides with roles in the production and release of root cap cells. Here, we review the involvement of the known players in root cap cell release, compare the release of border-like cells and border cells, and discuss the importance of root cap cell release to root health and survival. Unlike animals, plant cells are fixed and attached together at the cell wall. However, there are several developmental processes in which cell separation is required and critical for plant survival. These include development of intercellular air spaces, leaf abscission, and seed dehiscence ,2. AnothBrassica napus (rapeseed), Brassica oleracea (Brussels sprout), and Sinapis alba (Mustard), BCs are released from the root tip as an organized layer of several cells that remain attached and are called “border-like cells (BLCs)”  transcription factors including FEZ, SOMBRERO (SMB), BEARSKIN1 (BRN1), BRN2, ANAC046, and ANAC087 have important roles in root cap development and cell release. FEZ is preferentially expressed in root cap stem cells and promotes periclinal divisions in these cells  and fungal (chitin and fusaric acid) microbe-associated molecular patterns (MAMPs) activate defense responses by triggering the production of reactive oxygen species in BLCs of both Arabidopsis and flax . After emicrobes . Mutatinmicrobes ,65.Medicago truncatula BCs inhibits growth of the soilborne fungal pathogen Phymatotrichopsis omnivora, causal agent of cotton root rot [Nectria haematococca suppressed growth of the fungus [Metabolites secreted from BCs also may function in defense. For example, flavonoid 7,4′-dihydroxyflavone from root rot . Exudatee fungus .Aphanomyces euteiches, an oomycete pathogen of pea, were attracted to pea AGPs in a chemotaxis assay [A. euteiches zoospores appeared to be inhibited by pea AGPs [Solanum tuberosum) root exudate, enriched in galactose-containing molecules including arabinogalactan proteins, impacts the growth of bacteria Pectobacterium atrosepticum that causes stem wilting of potatoes [Arabinogalactan proteins (AGPs) are secreted from the cell walls of BCs and BLCs of several species ,26,67 anpotatoes .Glycine max L.), exposure to Al for 48 h promoted mucilage production in BCs [Phaseolus vulgaris), the viability of Al-resistant border cells exposed to 200 µM Al in an agarose gel was higher than that of border cells from an Al-susceptible cultivar [2+ toxicity result in decreased border cell viability in rice [2+ tolerant rice genotype, Fe2+-sensitive rice roots had fewer BCs after exposure to high levels of Fe2+ [Besides protection from biotic stresses, BCs have been reported to protect plant roots from abiotic stress. For example, BCs of different crops immobilize aluminum in order to protect the growing root ,68,69. In in BCs . Comparen in BCs . At highn in BCs . In snapcultivar . In pea, in rice . Compare of Fe2+ .Root cap cell release is developmentally programmed and influenced by transcription factors, small peptides, and hormones. While we are beginning to understand the molecular players involved in this process, many questions remain, including:How do external stresses impact the release of root cap cells and the molecular pathways that control release? How are such stresses communicated in the root cap such that root cap cell production and release remain linked during environmental stress? Do differences in either the timing or type (BLC vs BC) of release impact whole root growth and root architecture? Do BLC and BC release impact root cap protection differently?How are signals between the separating layer and stem cells communicated and what are additional signals?What are the mechanisms used by the auxin pathway to regulate cell production from CSCs and the release of BLCs?What other transcription factors are involved in BLC and BC release? Are there other hormones besides auxin involved in BLC release? Does WOX5 have an impact on BLC release?