The entwined regulation of ATP production and antioxidant systems within the RBC also exploits Hb-based O2-sensitivity to respond to various physiologic and pathophysiologic stresses. O2 offloading, for example, promotes glycolysis in order to generate both 2,3-DPG  and ATP. Alternatively, generation of the nicotinamide adenine dinucleotide phosphate (NADPH) critical for reducing systems is favored under the oxidizing conditions of O2 abundance. Dynamic control of ATP not only ensures the functional activity of ion pumps and cellular flexibility, but also contributes to the availability of vasoregulatory ATP that can be exported when necessary, for example in hypoxia or upon RBC deformation in microvessels. RBC ATP export in response to hypoxia or deformation dilates blood vessels in order to promote efficient O2 delivery. The ability of RBCs to adapt to the metabolic environment via differential control of these metabolites is impaired in the face of enzymopathies , blood banking, diabetes mellitus, COVID-19 or sepsis, and sickle cell disease. The emerging availability of therapies capable of augmenting RBC ATP, including newly established uses of allosteric effectors and metabolite-specific additive solutions for RBC transfusates, raises the prospect of clinical interventions to optimize or correct RBC function via these metabolite delivery mechanisms.Metabolic homeostasis in animals depends critically on evolved mechanisms by which red blood cell (RBC) hemoglobin (Hb) senses oxygen (O Hb and the RBC serve metabolic responsiveness not only in their function as mobile reservoirs for O2 and CO2, but also via the regulated release of vasoactive mediators. Such mediators include the central metabolite ATP and S-nitrosothiols (SNOs), which are derivatives of nitric oxide  (Red blood cells (RBCs) and their exquisitely evolved, oxygen-carrying hemoglobins (Hbs) allow multicellular organisms to respire y cycle) . Here wevia two competing branches  prevented the proadhesive effect of inhibiting ATP export  as one component in the release of ATP from RBCs (in vivo and into the supernatant in vitro (via adenosine receptors promotes deoxygenation of sickle hemoglobin and in turn (HbS) polymerization and RBC sickling. RBC ATP export is increased in hypoxia, and the resulting increases in plasma ATP in resistance vessels such as arterioles may contribute to hemodynamic responses to hypoxia. We recently demonstrated that in mice lacking the ATP exporter Px1, the hypotensive and hyperemic (blood flow-increasing) responses to hypoxia were significantly diminished, consistent with a role for ATP export in these responses  displayed greater temperature-dependent binding to ECs than “knobless” erythrocytes infected with the same pathogen. Rather than a simple physical advantage in cell–cell binding, the proadhesive effect of the presence of knobs may reflect a localized abundance of adhesive ligands and receptors  has been typically viewed as passive. RBC membrane disorders predispose to thrombosis , and folAn important consequence of the recently recognized capacity of RBCs to export antiadhesive mediators is that the exported molecules can modulate not only the stickiness of the cell of origin, but also of neighboring cells. We showed, for example, that coincubation of SCD RBCs with fresh healthy (AA) RBCs attenuated the adhesivity of the SCD RBCs, whereas coincubation of AA RBCs stored for 30 days did not attenuate SS RBC adhesivity .via regulation of permeability . We have discussed these important technical considerations in detail elsewhere, and recommended quality controls and a framework for interpretation and inter-laboratory comparisons. The centrifugation that is routinely used in order to separate RBCs from the other components of source whole blood, and to remove cell suspension buffers following application of pharmacological probes used to test mechanistic hypotheses, can itself deform the cells and elicit further, artifactual ATP release. In addition to eliciting deformation-induced release of mediators from RBCs, centrifugation can also promote the lysis of RBCs – particularly in the case of unhealthy RBCs – which also raises extracellular ATP levels and can be incorrectly ascribed to regulated ATP export.The mechanisms and consequences of export of vasoactive mediators from RBCs can be modeled  disease . Specifi disease , 2021. Win vivo, could liberate enough ATP to mimic (or mask) that resulting from regulated, non-lytic release, depending on such additional variables as hematocrit, PO2, and the presence of ectonucleotidases (ATPases)  ATP has been measured in the high-nanomolar to ∼1 μM range . NotablyATPases) . Interprvia interaction between Hb and the membrane-resident protein AE1 (aka Band 3)  residue of a protein . This reaction is allosterically controlled and coupled, in that the transfer of a NO derivative from a heme group in Hb to the Cys is favored in the oxygenated, “R” conformation of Hb . Convers Band 3) . The AE1 Band 3) . The fin Band 3) , 2007. O Band 3) .2 affinity, which in successive turn promotes HbS polymerization, RBC sickling, and hemolysis. The elevated plasma adenosine is believed to reflect both (a) increased plasma accumulation of ATP from RBCs themselves or tissues, and (b) activity of the ectonucleotidases CD39 and CD73, which hydrolyze ATP, leading ultimately to the formation of adenosine. Similar findings were demonstrated in mouse models of SCD and in blood from SCD patients. The effects of adenosine could be specifically inhibited by an antagonist of the A2BR adenosine receptor, suggesting a druggable process. Sickle RBCs are characterized by elevated indices of oxidative stress and depressed ATP , as wellRed blood cells from volunteers with glucose-6-phosphate dehydrogenase deficiency were significantly inferior to RBCs from G6PD-competent donors with respect to post-transfusion recovery , although the difference was small. This functional RBC storage lesion correlated with significant defects in glutathione , increased purine oxidation, and increased glycolysis (an expected shift in G6PD deficiency). When the G6PD inhibitor DHEA was used to decrease NADPH formation in RBCs, ATP export in response to deformation was also suppressed . Taken tn = 222) for patients with anemia (n = 1099), Hb levels were significantly lower in patients with severe illness than in those with non-severe COVID-19 disease. Modest but progressive declines in Hb values were statistically significant in ill COVID-19 patients, and morphologically abnormal RBCs such as stomatocytes and knizocytes were observed in the blood of COVID-19 patients  blood group antigens in COVID-19 and clinical outcomes suggests that the possibility of increased RBC adhesivity in this disease deserves further investigation  saw record numbers of cases over the last two years. The subsequent rush to biobank samples for molecular phenotyping has been unprecedented. Plasma and serum, which are easy to obtain, were the focus of dozens, if not hundreds, of ‘omic  studies of COVID-19 disease, including mass spectrometry-based proteomics and metabolomics . Changesvia limitation of the substrate glucose-6-phosphate. Systemic changes in metabolic control are also present in patients with COVID-19, and may persist well beyond the acute illness. Not only is diabetes an important comorbidity predisposing patients to COVID-19 infection, but insulin resistance was also detected in a substantial fraction of non-diabetic COVID-19 patients, even among normoglycemic patients , as reported in a multi-omic investigation . These mpatients . Moreovepatients .via the liberation of free heme, hemoglobin, and iron can be highly toxic)  or proliferation or dedifferentiation. In the anucleate RBC, there is still a substantial role for metabolic shifts in linking cell stress with either survival or with safe and accelerated clearance from the circulation. Specifically, RBCs rendered dysfunctional through nutrient, temperature, or osmotic stress can nevertheless avoid lysis (which y toxic) . Such cesubjects . While t2 delivery but also O2 consumption by the tissues  National Institutes of Health have recognized the need for updated approaches to the licensing of RBC products that incentivize the goal of improved clinical benefits in anemic patients receiving transfusion , a study endpoint chosen because of its adoption in clinical guidelines for trauma management including those for Advanced Trauma Life Support and from the American College of Surgeons  were non-inferior to fresher RBCs  in ICU and cardiac surgery patients among others , but thiSurgeons . The meti) implicated in transducing regulated ATP export  takes place at the expense of DPG. Patients with pyruvate kinase deficiency (PKD) are susceptible to hemolytic anemia  because 2) in leukoreduced RBC units obtained from each of several independent blood product suppliers and then stored in AS-3 additive solution , but also on the parallel CO2 elimination effected by the sparging (gas flushing) used to deoxygenate the RBCs. When RBCs were stored under isocapnic conditions under which CO2 concentration  was held steady, DPG declined at rates similar to those in conventionally stored RBCs  in sickle cell disease can safely convey a net clinical benefit . “Positivia reactive oxygen species) and its mission of efficient O2 delivery and CO2 clearance. Glycolysis generates ATP and DPG preferentially when RBCs have offloaded O2, coinciding with the O2 delivery limb of the RBC circuit, via both bulk O2 transfer and blood flow control. Disordered generation of these metabolites characterizes several health conditions and disease states. New and repurposed therapeutic approaches can drive the generation and export of ATP and are worthy of investigation.The regulated production of key RBC metabolites, including vasoregulatory ATP, the allosteric effector DPG, and the reductant NADPH, is sensitive to cellular cues unique to the RBC, namely its high oxidant load .TM conceived the manuscript. All authors participated in researching, writing, and editing the manuscript.TM received research funding from Hemanext LLC. A grant with Forma Therapeutics to TM is pending. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RGS2) is a gene involved in neuronal plasticity and synaptic signaling, whose expression in the brain is altered in neuropsychiatric and neurodegenerative disorders. Microarray data from large datasets suggested reduced RGS2 mRNA levels in the post-mortem brain tissue and blood of Alzheimer’s disease (AD) patients. The results were previously confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) only ex vivo in lymphoblastoid cell lines derived from AD patients and controls. In this study, we compared RGS2 mRNA levels in peripheral blood samples from 69 mild cognitive impairment (MCI) patients to 50 age- and sex-matched non-cognitively impaired controls, out of which 25 patients were monitored at 1 year. We found that RGS2 was indeed downregulated in the peripheral blood of these patients , and despite disease-specific therapy, RGS2 transcript levels continued to decrease at 1 year. The results suggest that RGS2 seems to be involved in AD pathology and progression and can be introduced in a panel of blood AD biomarkers.Regulator of G protein signaling 2 ( Mild cognitive impairment (MCI) is a neurological disorder characterized by a slight but noticeable decline in cognitive abilities, with minimal impact on daily living activities. MCI patients have an increased risk of developing Alzheimer’s disease (AD). It was recently shown that 20–40% of MCI individuals will progress to AD-related dementia within 3 years of the initial diagnosis  biomarkers that reflect pathologic aberrations in AD brains are currently used for AD diagnosis  mRNA levels as a potential biomarker for AD, showing (by a data mining approach) lower RGS2 mRNA levels in AD patients compared to non-aged-matched controls in several brain regions and blood  family, which attenuates signaling by heterotrimeric G proteins by selectively inhibiting Gqα function .The aim of this study was to extend previous findings indicating downregulated RGS2 mRNA levels were evaluated by qPCR on an ABI-7500 fast instrument (Applied Biosystems) with SYBR green chemistry. The assay used for RGS2 was designed on NM_002923. RGS2 expression levels were normalized on the geometric mean of two housekeeping gene transcripts, HPRT1 (NM_000194) and RPLP0 (NM_001002), whose stability in whole blood has been previously assessed  were recruited and diagnosed at the Hospital Universitari Santa Maria-IRBL Leida, Lleida, Spain, and at the Department of Neurology of the Hospital Clinico San Carlos of Madrid, Spain, according to the recommendations issued by the National Institute on Aging and Alzheimer’s Association . We further correlated RSG2 mRNA levels with age and, no correlation was observed in the whole group , in MCI patients , or in CTRL , highlighting that the changes in RGS2 levels are not age-dependent. We also correlated RSG2 mRNA levels with CSF biomarkers and no significant correlation was found for the 33 patients evaluated with the Innogenetics ELISA kit , nor for the other 36 patients investigated using the Lumipulse G600II instrument . Since RGS2 mRNA levels were not normally distributed , the differences between the MCI and CTRL groups were evaluated with the Mann–Whitney non-parametric test. We found that RGS2 levels were downregulated in the blood of MCI patients compared to controls . In particular, for 21 patients out of 25 a decrease of 66 ± 25% was registered , showing that the levels of RGS2 continued to decrease despite the treatment.The two groups of patients and CTRL were homogenous for sex, age, education level, and presence of comorbidities, except for hypercholesterolemia, which was more frequent in the MCI group . We firs disease . In the gistered , in paraRGS2 mRNA levels in the whole blood of MCI patients compared to CTRL, supporting previous results obtained from LCLs derived from AD patients, and microarray data from the blood of MCI and AD patients. We also observed a positive correlation between the MMSE score and RGS2 blood levels, as well as a significant decrease in this transcript during the progression of the disease.Our qRT-PCR study detected a 1.6-fold decrease in the RSG2 is an immediate-early gene, whose expression is rapidly induced in the neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity (Ingi et al., At neuronal synapses, RGS proteins together with G proteins and GPCRs, act in regulating neurotransmitter release, down-stream signaling, synaptic transmission, and synaptic plasticity (Gerber et al., RGS2 gene have been associated with different neuropsychiatric diseases such as anxiety disorders across multiple levels of assessment (childhood temperament, adult personality, and brain function; Smoller et al., RGS2 was associated with post-traumatic stress disorder under conditions of lifetime exposure to a potentially traumatic event and low social support (Amstadter et al., RGS2 polymorphic variants may experience differential affective responses to smoking tobacco, which could make them vulnerable to developing nicotine addiction (Rorabaugh et al., RGS2 seem to predict the severity of schizophrenia symptoms (Campbell et al., RGS2 has also been associated with attention deficit hyperactivity disorder (ADHD; McCaffrey et al., Genetic variants within the RGS2 expression was found in Huntington’s disease patients (Seredenina et al., RGS2 mRNA levels in the striatum of rats undergoing dopamine depletion suggested a role in Parkinson’s disease (PD; Geurts et al., RGS2 has been indicated as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients (Dusonchet et al., RGS2 polymorphisms and antipsychotic-induced Parkinsonism have been evidenced (Greenbaum et al., Regarding neurodegenerative diseases, decreased striatal RGS2 in AD has been conducted thus far, showing that RGS2 mRNA levels have a 3.3-fold lower expression in AD LCLs compared with controls. Moreover, lower RGS2 expression levels were found in public gene expression datasets from post-mortem MCI and AD brain tissues compared with controls in the posterior cingulate, superior frontal gyrus, medial temporal gyrus, and in the blood (Hadar et al., A single study investigating the role of RGS2 mRNA levels in the blood of MCI patients, as compared to controls, confirming and validating previous findings through qRT-PCR (Hadar et al., RGS2 blood levels decreased during disease progression. Altogether, our study suggests that RGS2 might be a valuable blood biomarker in AD that has the potential to be implemented for clinical use if included in a panel of multiple biomarkers.In conclusion, our results evidenced decreased The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Hospital Arnau de Vilanova de Lleida, Lleida, Spain—CE 1218 and Hospital Clinico San Carlos—19/141 ethics committees. The patients/participants provided their written informed consent to participate in this study.EM designed and coordinated the study, performed the statistical analysis, and wrote the first draft of the manuscript. GP-R and JM-G were responsible of the clinical evaluation and the samples collection. MD and CC performed the laboratory experiments, contributed to data processing, and writing. GM and AC gave their contribution in data interpretation, writing and critical reading, and revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height. Defining the molecular and cellular mechanisms underlying bone development is essential for getting a better understanding of human skeletal diseases . EndochoXLH OMIM #307800) is the most common inherited form of rickets, with an estimated frequency of 1 in 20,000 patients 07800 is ,5. OtherXLH is an inherited disease of phosphate metabolism caused by inactivating mutations of the phosphate-regulating endopeptidase homolog X-Linked (PHEX) gene ,6,7. Mor2D). FGF23 has been reported to be responsible for hypophosphatemia and reduced 1,25-(OH)2D levels in animal models  contribute to many symptoms of XLH , an active vitamin D3 analog approved for osteoporosis therapy in Japan, is a more active stimulator of calcium or Pi absorption in normal rat and mouse intestines compared with 1,25D; however it causes FGF23 resistance in Hyp mice similar to conventional treatment . AlthougThe use of GH in patients with XLH leads to a significant increase in linear growth and height, as well as a normalization of PTH levels, an increase in calcitriol levels, and a reduction in renal phosphate excretion. However, the main complications of this treatment are skeletal deformities and body disproportion due to the failure in correcting the GP abnormalities, as it might implicate a higher growth in some zones compared to others ,66. In tMaintained treatment with supplements of phosphate and 1-OH hydroxylated vitamin D metabolites in XLH children accelerates longitudinal growth velocity, but does not fully correct the final height reduction, even when the osseous lesions of active rickets are healed. Consistent alterations in growth hormone (GH) secretion are not present in patients with XLH.The standard treatments for XLH patients are based on prescribing oral phosphate and calcitriol. However, it does not completely rescue the rickets, nor does it alleviate all of the associated symptoms. Serious side effects such as nephrocalcinosis and hyperparathyroidism have also been observed. Novel therapies inhibiting the excessive activity of FGF23 or replacement therapy with recombinant FGF23 may be beneficial for those patients with abnormal GP development and several metabolic bone diseases, including XLH . Kaneko 3 levels and normalizing growth plate thickness [Treatment with a FGF23-neutralizing antibody (FGFG23Ab) from four to eight weeks normalized the femoral ash weight, GP thickness and serum Pi levels as well as increasing circulating 1.25(OH)2Dhickness . This dihickness ,64,73. Bhickness . On the Dr. Santos’ Laboratory demonstrated that inhibiting the MAPK pathway (FGF23 downstream pathway) in PHEX mice partially rescues growth impairment by normalizing the GP structure, specifically in the hypertrophy zone . The C-tX-linked hypophosphatemia is a skeletal disorder characterized by several skeletal features, the main ones being growth retardation, osteomalacia in adults and rickets in children. A better understanding of the alterations in the dynamics and maturation of the GP seems to be the key for improving the growth and bone phenotype. Furthermore, newly developed therapeutic strategies targeting FGF23 have shown promising short-term results in normalizing growth and decreasing the severity of rickets. Nevertheless, further studies analyzing the effect of a long-term treatment and the impact on adult height are needed, and seem to be the next step to further determine the effectiveness of the treatment. The cartilage in general, and the GP in particular, are tissues that are hardly approachable in vivo, and they justify the use of animal models such as the Hyp mouse.The mechanism by which FGF23 inhibition affects GP and cartilage dynamics needs to be clarified, as well as the predisposition to develop OA, especially in infants; how this is translated into a growth rescue, and whether FGF23 related treatments would be suitable for pediatric patients, also needs clarification. In the same way, it would be attractive to know how the treatments (FGF23 blockers) affect the predisposition to develop OA . Further


The central dogma of RNA biology was traditionally governed by the transcription of DNA into messenger RNA (mRNA) followed by translation into protein. As additional functions of RNA were discovered, it became clear that there were many species of RNA, and many of those species were multifunctional with roles outside of coding for protein. One such area of discovery was the ability to form circular RNAs (circRNAs) that have been shown to be important in a wide range of cellular processes and as such are important in health and disease. These functions broadly include the modulation of gene expression, facilitation of protein–protein interactions and protein translation. It is important to understand how the dysregulation of these processes are involved in cancer progression and the avenues this may open for the targeting or overexpression of circRNAs as novel therapeutics.The majority of RNAs transcribed from the human genome have no coding capacity and are termed non-coding RNAs (ncRNAs). It is now widely accepted that ncRNAs play key roles in cell regulation and disease. Circular RNAs (circRNAs) are a form of ncRNA, characterised by a closed loop structure with roles as competing endogenous RNAs (ceRNAs), protein interactors and transcriptional regulators. Functioning as key cellular regulators, dysregulated circRNAs have a significant impact on disease progression, particularly in cancer. Evidence is emerging of specific circRNAs having oncogenic or tumour suppressive properties. The multifaceted nature of circRNA function may additionally have merit as a novel therapeutic target, either in treatment or as a novel biomarker, due to their cell-and disease-state specific expression and long-term stability. This review aims to summarise current findings on how circRNAs are dysregulated in cancer, the effects this has on disease progression, and how circRNAs may be targeted or utilised as future potential therapeutic options. Cancer affects approximately 5% of the population in Western countries and, despite an increasing five-year relative survival rate in the last decade, still accounts for ~14.6% of all human deaths . FurtherCircular RNAs (circRNAs) are a subset of ncRNAs, characterised by their closed loop structure, that have recently been identified as important factors in the development of various diseases. The abundant and dynamic nature of circRNA expression has highlighted the capability of circRNAs to be involved in many molecular processes. In particular, circRNAs have been shown to be aberrantly expressed in a multitude of different cancers and furthermore can be involved in the regulation of tumourgenesis through RNA sponging, protein interaction scaffolding, mRNA transcription regulation and protein translation . FurthercircRNAs are structurally defined by a characteristic covalently closed loop structure that is primarily derived through the backsplicing of canonical splice sites . FlankinIntronic circRNA genesis follows a broadly similar mechanism, with circRNA formation promoted through proximal intron splice sites, forming a lariat intron. Subsequent evasion of debranching and degradation allows the 3′ tail of the RNA lariat to be removed increasing stability of the circRNA .circRNAs have the potential to act as key competitive endogenous RNAs (ceRNAs) of miRNAs and other small regulator RNAs, and as such may be crucial regulators in cancer. Research has previously focused on the direct interactions between miRNAs and target mRNA dysregulation in cancer, however, the interactions between ncRNAs regulating these processes further upstream is still relatively unexplored.Small regulatory RNAs are a class of RNAs that include endogenous microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). miRNA biogenesis involves the cleavage of imperfect ~33 nt RNA hairpins into pre-miRNA stem-loops by Drosha and associated co-factors forming the Microprocessor complex  11]. Al. Al11]. FAK), Insulin Like Growth Factor 1 Receptor (IGF1R), Epidermal growth factor receptor (EGFR) and Yin Yang 1 (YY1) resulting in increased proliferation, EMT and migration. Upregulation of ciRS-7 has been found in a range of cancers including gastric, lung and colorectal [The regulation of miRNA activity can occur via the binding of miRNAs to a miRNA “sponge”. This sponging activity has been shown to be an essential function of some circRNAs . In ordelorectal . Additiolorectal . There hMBL) pre-mRNA for the spliceosomal machinery. Conserved MBL protein binding sites are found on the flanking intronic sequences of circMBL. Therefore MBL can stimulate circRNA biogenesis through binding and bridging of flanking introns containing MBL binding sites- including its own circMBL and decreasing mRNA levels [Transcriptional regulation of circRNA begins with the biogenesis of circRNAs and its fundamental co-transcriptional nature. There is a negative correlation between linear and circRNA expression when the majority of exons are circularised, suggesting competition between the linear and the circular transcripts for the spliceosomal machinery . TypicalA levels . FurtherA levels .An emerging area of study is the interaction between circRNAs and RNA binding proteins . These iFLI1 gene in the development of breast cancer occurs via the FECR1 circular RNA interacting with the FLI1 promoter through extensive demethylation, promoting metastasis [A further mechanism of circRNA regulation of transcription occurs via induction of DNA hypomethylation in the promoter region of the parental gene or by regulating intronic enhancers. The regulation of the tastasis .Although classified as ncRNAs, there is evidence that certain circRNAs are able to be translated under certain conditions, although this is still controversial. circRNAs have in proof-of-principle experiments been shown to lead to translation of protein, and this may occur naturally in tissues that have higher levels of circRNA expression, namely muscle and neuronal . SeveralOther roles of circRNAs are mediated via protein interactions, including but not limited to sponging, forming scaffolds or decoys. circRNAs due to their covalently closed ring structure have the potential to form different RNA 3D structures compared to their linear transcripts, in turn potentially leading to completely different binding partners and novel interactions . As a keDNMT1) and recruits the Tet Methylcytosine Dioxygenase 1 (TET1) protein, TET1, through its demethylase activity, demethylates the CpG regions of the DNMT1 promoter, resulting in decreased DNMT1 transcription and therefore decreased protein levels, leading to demethylation of FLI1 DNA, enhancing its expression. Through the binding and recruitment of TET1, circFECR1 has the potential to be a master regulator in breast cancer, aiding in oncogenesis.Through these protein interactions, circRNAs are capable of widespread cellular dysregulation, for instance through epigenetic-mediated regulation of oncoproteins. As mentioned previously, the proto-oncoprotein FLI1 has been found overexpressed in breast and lung cancer, correlating with a poor prognosis. FLI1 overexpression has been linked to the function of a novel circRNA: circFECR1 . circFECDNMT1 mRNA, thereby leading to increased DNMT1 stability and therefore increased translation of DNMT1. Increased levels of DNMT1 enhance p53 promoter methylation in turn decreasing p53 transcription and aiding tumour proliferation and survival [A further key example of circRNA-protein interactions contributing towards oncogenesis is circDNMT1 expression in breast cancer. Cells with high levels of circDNMT1 have a higher senescence induction tolerance, allowing further replicative cycles and therefore promoting tumuorigenesis. Mechanistically, circDNMT1 binds to the protein Auf1. circDNMT1 through its binding to Auf1 leads to its nuclear translocation, preventing its usual cytoplasmic binding to survival .circRNAs can conversely protect against and negatively regulate oncogenesis through specific protein interactions. circZKSCAN1 levels are reduced in hepatocellular carcinoma (HCC) through the activity of QKI5 and this reduction correlates with poorer survival rates. circZKSCAN1 binds to the protein FMRP acting as a competitive inhibitor, which prevents FMRP activating the downstream target CCAR1 which in turn prevents CCAR1 mediated activation of Wnt/β-catenin . FurtherThe role of chromosomal translocations leading to the production of fusion gene products is well-known phenomenon in cancer, with examples including BCR-ABL and PML-RARA  with theExosomes are extracellular vesicles averaging around 40–160 nM in diameter, surrounded by a lipid bilayer. Discovered in the 1980s, their original role was thought to be restricted to cellular waste disposal. Since then, however, they are thought to function as key cellular messengers, transporting molecules including proteins, mRNA and metabolites all around the body . As partcircRNAs have the potential to be key components of exosomes due to their inherent stability and multiple regulatory roles, with initial studies identifying the contents of tumour cell exosomes to vary from those of a healthy individual. Of particular note is the ratio of circRNAs to their linear counterparts in the tumour cell exosomes, which was up to six-fold higher, suggesting higher levels of incorporation against the natural cellular circBiome and therefore specific selection . FurtherAlready there is functional analysis of specific exosomal enriched circRNAs in cancer, one study identified circPSMA1 as a circRNA enriched in cancer cell derived exosomes. Functional analysis showed this circRNA acted as a miRNA sponge, targeting the tumour suppressive miR-637, in turn leading to increase in Akt1 expression, and increases in β-catenin and cyclin D1 . Due to circRNAs in exosomes can also aid in oncogenesis through mechanisms outside the primary hallmarks of cancer, with roles identified in the emerging hallmarks, namely glucose metabolism. Cancer cells have increased metabolic activity, and advanced cancer is often associated with cachexia. It is theorised this state can be due to increased levels of brown adipose tissues (BATs) formed from white adipose tissues (WAT)  which leDue to the ability of exosomes to migrate and play key roles as messengers, exosomes can particularly contribute to metastasis. Thereby, exosomal circRNAs also have the potential to aid in the formation of metastatic tumours, with the original primary tumour secreting pro-oncogenic circRNAs into exosomes to help prime a pre-metastatic niche in secondary locations around the body. A clinical example of these exosomal circRNAs playing a role in promoting metastasis would be circIARS. circIARS was found to be upregulated in pancreatic cancer patients’ tissues and furthermore enriched in exosomes found in the blood plasma. Higher levels of circIARS correlated with a poorer prognosis in patient survival and increased chance of metastasis from the primary tumour. Further experiments found exosomal circIARS was taken up by HUVEC cells at secondary sites, which led to increases in the permeability of the endothelial cells, aiding in the formation of metastatic sites. circIARS, once at the secondary sites, binds miR-122, in turn leading to increased levels of RhoA, increased F-actin and decreased ZO-1 to aid in endothelial cell monolayer permeability, thereby aiding metastatic spread . FurtherThe potential of circRNAs as biomarkers and therapeutics has gained significant interest in recent years due to their stability and specificity to certain diseases. Due to their closed structure, circRNAs are resistant to many cellular RNA decay mechanisms. Many studies have identified circRNAs to be specifically dysregulated or enriched in specific cancers. For instance, circPDE8A was found to be upregulated in exosomes released from pancreatic cancer cells, and through sponging of miR-338, it leads to the activation of MACC/MET/ERK and AKT pathways promoting invasion . Of partThis excretion of circRNAs into exosomes is of particular interest if these circRNAs are excreted into the serum, plasma and blood, allowing non-invasive detection. Studies have already identified that circRNAs can be highly expressed in the saliva and the blood, allowing relatively non-invasive collection. Moreover, coupled with specificity for specific diseases and high stability, circRNAs have the potential to be ideal clinical biomarkers ,49. circPTGR1 gene, with increased levels in all three correlating with a poorer prognosis in HCC patients [Already, levels of specific circRNAs correlate with severity of the disease and even survival rates. Three circRNAs are derived from the patients . While ipatients . InteresGiven circRNA specificity in established cancers and metastatic tumours, there is also the potential for circRNA biomarkers to be used in early detection during pre-malignant disease states. For instance, several circRNAs have been found to be dysregulated in colorectcal adenoma that could act as predictive biomarkers for transition to colorectal cancer (CRC) . AdditiocircRNA expression patterns have also been shown to be modulated under chemotherapy, with numerous instances of differences between circRNAs in chemoresistant compared to chemosensitive cells, from lung, to leukaemia and pancreatic . HoweverAdditionally, utilising circRNAs ability to sponge miRNA as a means of downregulating signalling pathways involved in cancer has been investigated as a novel therapeutic approach. miRNA sponges can be expressed as miRNA sponge cassettes that are formed of tandem repeats of identical miRNA targeting sites. These cassettes sequester endogenous cellular miRNAs, impairing the binding of the miRNA to the natural target, thus inhibiting the miRNAs post transcriptional regulation activity . AdvanceA final aspect of circRNAs therapeutic approaches is the potential to act as vectors for protein expression to aid in the treatment of disease. Currently, the majority of research is focused on the use of mRNA. However, some of the major hurdles to using mRNA include its natural instability and ability to trigger an immune response . EvidencThe study of circRNA dysregulation in cancer is an emerging field that offers exciting new avenues of research. The multitude of functions that circRNA perform within cellular biology has shown the breadth of dysregulation that can occur in many cancers and other disease states. Evidently, these functions are complex and intertwined and have a profound effect on signalling pathways, transcription and translation when dysregulated. Many questions remain concerning circRNAs, including the causal factors of dysregulation and how big a role they may play in driving oncogenesis. The potential to utilise the intrinsic stability and specificity of circRNAs for diagnostics and treatment in particular merits further investigation.


This body of work may serve to furnish existing interest while also constituting a springboard of sorts for indispensable further investigation on neuroimmune interactions in chronic pain. The growing awareness of the critical role played by the innate and adaptive immune systems in the mechanisms underlying chronic pain has prompted further research examining the modalities by which immune cells communicate with neurones, the aim being to identify new players involved in inflammatory and neuropathic pain signalling. This collection of research includes 9 articles on neuroimmune interactions as the underlying mechanisms for neuropathic pain including peripheral neuropathies, pain in rheumatoid arthritis and osteoarthritis, and bone cancer pain. The immune cells under scrutiny include macrophages, microglia, osteoclasts, and B cells, and their interactions with neurones in locations such as the dorsal root ganglia, blood–spinal cord barrier, spinal cord, and brain. Our hope is that this body of work may serve to furnish existing interest while also constituting a springboard of sorts for indispensable further investigation on neuroimmune interactions in chronic pain. The loss of this protective mechanism has considerable physical consequences that interfere with quality of life. However, more commonly, pain outlives its usefulness and becomes chronic pain, which is profoundly different from acute pain as it could stem from plastic changes within the pain pathways.3Acute pain perception involves specialised sensory neurones—namely nociceptors—which transmit noxious signalling to dorsal horn neurones in the spinal cord. From here, the transmission ascends to higher centres in the brain. It is in the brain that the intensity and type of pain is perceived, the emotional component is registered, and followed by protective actions in an effort to safeguard the body's integrity.Chronic pain can be a debilitating condition, in some cases it lasts longer than the initial stimulus and for which the pain experienced is disproportionate to its instigator. Chronic neuropathic pain, such as that in peripheral neuropathy, and chronic musculoskeletal pain, such as that in osteoarthritis and rheumatoid arthritis, can be characterized by an increase both in sensitivity to painful stimuli  and in painful response to nonnoxious stimuli .13Under sensitised pain conditions local inflammatory reactions—for instance, through macrophage accumulation and cyto (chemo) kine production—facilitate the sensory neurones' activation and contribute to peripheral sensitisation. The increased afferent input into the dorsal horn of the spinal cord, remotely from the injury, causes increased neuronal activity  and significant responses by resident immune cells—namely microglia—which further promote neurone activity by releasing pronociceptive factors.9The growing awareness of the critical role played by the innate and adaptive immune systems in the mechanisms underlying chronic pain has prompted further research examining the modalities by which immune cells communicate with neurones, the aim being to identify new players involved in inflammatory and pathological pain signalling.This collection of research includes 9 articles on neuroimmune interactions as the underlying mechanisms for neuropathic pain including peripheral neuropathies, pain in rheumatoid arthritis and osteoarthritis, and bone cancer pain. The immune cells under scrutiny include macrophages, microglia, osteoclasts, and B cells, and their interactions with neurones in locations such as the dorsal root ganglia (DRG), blood–spinal cord barrier, spinal cord, and brain.7 within their well-received work on the P2X4 receptor that is de novo expressed by dorsal horn microglia after peripheral nerve injury. P2X4 activation by adenosine triphosphate results in the release of brain-derived neurotrophic factor that, through neuronal TrkB receptor activation, generates a decrease in the potassium/chloride transporter and leads to a depolarising shift in chloride ion reversal potential, which ultimately results in gamma aminobutyric acid being less inhibitory. This review offers a critical perspective on the suitability of P2X4 receptor blockers for the treatment of chronic pain and the role of microglial activation in human chronic pain conditions.The role of microglial-mediated mechanisms in neuropathic pain  is thoroughly covered by Khono and Tsuda4 introduce the low density lipoprotein receptor-related protein 1 (LRP-1) as playing a potential protective role in neuropathic pain: for instance, the activation of LRP-1 in microglia results in a decrease of proinflammatory cytokine expression. This comprehensive review examines LRP-1 and LRP-1 ligands potentials in the treatment of neuropathies.Garcia-Fernandez et al.2 discuss the role of components within the renin–angiotensin system in neuropathic pain, placing emphasis on the octapeptide angiotensin II (Ang II) and modulators of G-protein-coupled receptors ATR1 and ATR2, which are expressed in pain-related areas of the brain by neurones, microglia, and astrocytes. They explore and evaluate evidence for the efficacy of AT2R antagonists in neuropathic pain preclinical and clinical work. Their own data demonstrate that AT2R activation by Ang II in macrophages contributes to nociceptive hypersensitivity in neuropathic pain models. Macrophages are found in significant numbers at the site of nerve injury, and Ang II facilitates monocyte/macrophage infiltration into said site and triggers production of radical oxygen species that can activate TRPA1 receptors on sensory neurones to induce nociceptive signalling.Balogh et al.1 explore the mechanisms underlying bone cancer pain, which shares feature with neuropathic and inflammatory pain and results from breast and prostate cancers metastases to the bone, a tissue that is richly innervate by DRG nociceptors. Here, the innate immune cells scrutinised are bone-resorbing osteoclasts, whose differentiation is accelerated by tumour cells and are found in close vicinity to the nociceptor peripheral terminals. Osteoclasts contribute to pain by producing proinflammatory cytokines and chemokines, by creating an acidic extracellular environment, and also indirectly by increasing bone resorption that weakens the bone and leads to nerve compression and fractures. Osteoclastogenesis provides scope for innovative antinociceptive targets that can be combined with the use of bisphosphonates for bone cancer pain relief. Most relevant to this special issue is the mention afforded to inhibitors of colony-stimulating factor 1 receptors (CSF1R) that may be considered for the reduction of pain in bone cancer models on the basis of preclinical evidence.Meanwhile, Andriessen et al.14 that supports the existence of a role for CSF1 and CSF1R in neuropathic pain, with specific attention on sensory neurone-derived CSF1, which is de novo expressed in injured neurones downstream of transcription factor ATF-3. Extracellular CSF1 acts on CSF1R expressed by microglia in the spinal cord and resident macrophages in the DRG and contributes to these cells' acquisition of a pronociceptive phenotype as well as the establishment of neuropathic allodynia. Moreover, CSF1 mediates dorsal horn microglial proliferation after peripheral nerve injury to a significant extent—this is an important contribution that deserves further investigation considering microglia are self-renewal cells and monocytes do not seem to infiltrate the spinal cord after peripheral nerve injury. The latter topic is covered in greater depth by Montague-Cardoso and Malcangio10 who examine existing evidence suggestive of a possible breach of the blood–spinal cord barrier in models of neuropathic and inflammatory pain. This breach, when established, generates monocyte/macrophage infiltration and the establishment of pronociceptive communication with dorsal horn neurones alongside the microglia.Indeed, there is convincing evidence reviewed by Yu et al.14 also maintain that DRG tissue macrophages play a significant role in the induction of neuropathic allodynia and display a similar role in both male and female mice. The authors favour the possibility that DRG resident macrophages are a self-maintained population that proliferates after peripheral axon injury. What is more, they discuss a pivotal role for neurone-derived CSF1 in macrophage proliferation in male mice only.Yu et al.11 are in agreement with the emerging suggestion that DRG resident macrophages are a self-maintained population that proliferates and contributes to neuropathic allodynia after nerve injury and chemotherapy drug treatments. They propose that the activation of toll-like receptors  and nucleotide-binding oligomerization domain-like receptors in macrophages results in the production of cytokines and radical oxygen species that exacerbate nociceptive signalling. Alongside damage-associated molecular patterns such as the high-mobility group box-1 released by neurones, the suggestion is that pathogen-associated molecular patterns derived from gut microbiota may reach the DRG through the circulation and activate resident macrophages.Meanwhile, Silva et al.5 with a view to exploring new avenues towards novel interventional strategies. In this review, we find interesting comparisons of synovial macrophage phenotypes in OA and rheumatoid arthritis (RA) joints in both preclinical and clinical samples. The overall submission is that the profiling of macrophages may predict OA disease severity and pain, and that macrophage-associated pathways may provide a source of targets for OA pain. In animal models of OA, growing evidence indicates that synovial macrophages, DRG macrophages, and microglia in the spinal cord contribute to the underlying pain mechanisms. This comprehensive review article will stimulate more work on neuroimmune interactions in OA pain.The role of macrophages in the pathogenesis of osteoarthritis (OA) joint pain and their communication with nociceptors in the joints is considered by Geraghty et al,8 ask how autoantibodies contribute to chronic pain mechanisms in autoimmune diseases. Here, the adaptive immune cells under scrutiny are pathogenic B cells that release cytokines or autoantibodies and interact with the nociceptor peripheral terminals  and cell bodies in the DRG. The critical point made in this final review is that pain in autoimmune diseases can be present before clinical signs of inflammation, and emerging evidence indicates that immunoglobulins (IgG and IgM) can promote persistent pain states. IgG and IgM antibodies activate complement components that are pronociceptive through local cytokine production that sensitises nociceptive neurones. In addition, the Fc region of IgG can interact with Fcγ receptors expressed by immune cells (including microglia) and sensory neurones, which, after activation, can promote several events leading to pronociceptive effects.Remaining in the area of RA, with the addition of complex regional pain syndrome and channelopathies from potassium channel complex autoimmunity, Lacagnina et al.To conclude, I trust this body of work serves to furnish existing interest while also constituting a springboard of sorts for indispensable further investigation on neuroimmune interactions in chronic pain.The author has no conflicts of interest to declare.


The development and repair of dentin are strictly regulated by hundreds of genes. Abnormal dentin development is directly caused by gene mutations and dysregulation. Understanding and mastering this signal network is of great significance to the study of tooth development, tissue regeneration, aging, and repair and the treatment of dental diseases. It is necessary to understand the formation and repair mechanism of dentin in order to better treat the dentin lesions caused by various abnormal properties, whether it is to explore the reasons for the formation of dentin defects or to develop clinical drugs to strengthen the method of repairing dentin. Molecular biology of genes related to dentin development and repair are the most important basis for future research. Dentin constitutes the main body of the tooth and is located in the inner layer of enamel and cementum, the side wall of the pulp cavity and root canal. Dentin acts as a barrier to prevent exposure of the dental pulp, reduce the conduction of hot and cold stimuli to the pulp, and protect the living pulp. In addition, it also provides a hard tissue foundation for later dental restoration.1 Although the function of odontoblasts is unique, studies have not advanced new information likely because they are end-stage differentiated cells that are difficult to isolate and culture. To study the dentin formation process, it would be necessary to study gene regulation during the development and migration of odontoblasts.Odontoblasts are the only known cells that form dentin, and they are involved in the synthesis of primary, secondary, and reactionary dentin. A recent study showed that odontoblasts activate the innate immune response in the pulp.In this review, we present information on 300 genes involved in dentinogenesis  and to dExtracellular matrix (ECM) proteins could be directly involved in the construction of dentin because the mutation, deletion, or abnormal regulation of these proteins often leads to serious dentin-related diseases. Presently, all ECM proteins have been identified; however, their functional regulation still needs to be comprehensively studied. Furthermore, new regulatory mechanisms and key regulatory molecules are still being discovered.COL1) gene mutations(COL1A1 and COL1A2)4 are the main cause of this disease.Osteogenesis imperfecta (OI) with systemic skeletal dysplasia is a genetic systemic connective tissue disorder involving the bone, dentin, sclera, ear, blood vessels, skin, and other tissues. Type I collagen (9 Bone- and odontoblast-expressed gene 1 (Bono1) is a gene expressed in functional odontoblasts that is associated with regions of matrix mineralization.31 BSP may play an important role in the formation and mineralization during dentinogenesis.9Bone and dentin have similar mineralization processes, during which osteoblasts and odontoblasts first secrete unmineralized matrices termed osteoid and anterior dentin, respectively. The organic component of osteoid and anterior dentin consists of an ECM primarily composed of COL1 and several non-collagen proteins that play essential roles in the mineralization of collagen fibers during the conversion of osteoid and anterior dentin to bone and dentin, respectively.6 and is the first direct target of distal-less homeobox 3 (Dlx3), which was identified in odontoblasts.7Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are encoded by dentin sialophosphoprotein (Dspp) and its deficiency may lead to dentin hypoplasia or dysplasia and opalescent, shell, and abnormal deciduous teeth with short roots. Therefore, Dspp is an important marker of odontoblasts8 As a nucleus, bone sialoprotein (BSP) promotes crystal formation and in developing teeth it is well documented to form acellular cementum and periodontal attachments. However the expression and function of BSP in odontoblasts and dentin are uncertain.9 Dentin matrix acidic phosphoprotein 1/2 (Dmp1/2) participates in odontoblast differentiation and dentin formation.11 Rat Dmp3 is a compound protein of rat DSP and phosphophoryn.12 The formation of Dmp4 affects the growth and arrangement of hydroxyapatite crystals and promotes the differentiation of osteoblasts, ameloblasts, and odontoblasts.13In addition, TM14 has significant roles in the differentiation and maintenance of odontoblasts, and in dentin formation.18–20Matrix metalloproteinase 1 (MMP1) regulates tooth agenesis; MMP2 causes multiple cleavages near the DSP C terminus, releasing larger forms of DGP; and MMP8 may be involved in the alteration of dentin matrix during the development of human and rat tooth germ. In addition, MMP9 is important for tooth development and targets DSP during dentinogenesis, whereas MMP14 functions in tooth root formation, dentinogenesis, and tooth eruption. Furthermore, MMP20 cleaves DSP-DGP to generate DSP and DGP, and MMP1, 2, 9, 10, 11, 13, 14, 15, 16, 17, 19, 20, 23, 24 and 25 are expressed in odontoblasts.149 In dental attrition, hardened molds and highly mineralized layers were detected in sclerotic dentin. During the process of sclerotic dentin formation and long-term aging, the MMP content in dentin changes.150 The process of dentinogenesis is similar to bone formation and many genes are involved in both processes.The dentin organic matrix is secreted by odontoblasts during dentinogenesis and mineralized in the predentin area.32 Runx2 is also essential for the differentiation of osteoblasts and odontoblasts, and it regulates the expression of numerous bone- and tooth-related genes. Runx2 determines the lineage of osteoblasts and odontoblasts in the mesenchymal cells. Osterix (OSX), a downstream gene of Runx2 in the osteoblast differentiation signaling pathway, is involved in the differentiation, maturation, and intercellular signal transduction of odontoblasts.41 Osteoprotegerin (OPG) is expressed in both the thickened and bud epithelium, as well as in combination with receptor activator of nuclear factor-κΒ (RANK) in both the enamel and papillary stroma. Although RANK ligand (RANKL) was not detected in the tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells near the developing tooth germ.46Runt-related transcription factor 2 (Runx2) participates in dentin formation, mineralization, and the development of odontoblasts.Followed by the pre-section, we reviewed some key proteins related with odontoblasic differentiation or dentinogenesis. In further, the mineralization related pathways those could upregulate the mentioned proteins also can affect odontoblastic differentiation.47 TIMP1 plays a crucial role in the formation of tertiary dentin.The Wnt signaling pathway could currently be considered the most thoroughly studied pathway and drug research related to dentin regeneration has also achieved significant breakthroughs recently. However, the involvement of the Wnt non-classical signaling pathway in the dentin formation process has not been elucidated. This pathway plays a crucial role in the reactivation of immature pulp cells for tertiary dentinogenesis.47 Wnt signaling is essential to both the epithelium and stroma, whereas heterologous inactivation of β-catenin leads to stagnation of early tooth germ development. The elimination of mesenchymal-specific β-catenin stagnated tooth germ development, which indicates that Wnt signaling plays a role in the transition from the bud stage to the cap stage during tooth germ mesenchymal development. 50In addition, cavity preparation may activate the Wnt/β-catenin pathway.56 The role of the Wnt/β-catenin signaling pathway in odontoblast differentiation and dentin formation is mediated through its component proteins.56 Phenotype-related diseases are sometimes caused by paralog genes, which may explain the dental abnormalities in patients with Wnt10a and Wnt10b mutations.57 Wnt signaling is essential for normal tooth development and its persistent activation leads to the continuous renewal of teeth and supernumerary teeth, whereas its inhibition stagnates tooth development. Abnormal Wnt signaling has been shown to cause a variety of human developmental disorders ranging from the lack of a tooth to life-threatening cancer.57Wnt10a was shown to be strongly expressed in the odontoblast layer and was specifically expressed in the secretory odontoblasts co-expressed with DSPP.61 SHH is mainly expressed in the dental epithelium during tooth development.64Sonic hedgehog signaling molecule (SHH) has been shown to regulate dentin formation mainly during embryo development. GLI family zinc finger 1 (Gli1)-positive cells in mature teeth have been found to have stem cell properties that contribute to the regeneration of pulp and periodontal tissue. SHH, a secreted protein that plays a significant role in mammalian embryogenesis, regulates growth and determines the tooth shape. Dental epithelial SHH regulates tooth morphogenesis through epithelial mesenchymal signal transduction. Many studies have analyzed the function of SHH signaling in different stages of tooth development, and have reported that it regulates the formation of various tooth components, including the enamel, dentin, cementum, and other soft tissues.151 This delicate cell group has undergone an amazing process of evolution and degradation, which is closely related to the process of development from the dental cusp to the cervix. SHH is one of the signaling molecules for which the intermediate layer may play a role in mediated its function.151 The primary cilia are essential for the integration of Wnt and Hh signals and in their functional absence, SHH signals decrease in the dental stroma, whereas those of Wnt increase in the dental mesenchyme.67The stratum intermedium is a highly dynamic and SHH-expressing structure that undergoes marked and transient changes in the histological organization and phenotype during odontogenesis. The stratum intermedium is involved in the development of the tooth germ, which has not been previously reported.The transforming growth factor β (TGFβ) signaling pathway is mainly involved in the differentiation and regulation of odontoblasts and includes the BMP family of molecules, which showed the most remarkable effects.68 BMPs are signaling molecules secreted by the TGF superfamily and more than 30 types are known to regulate embryonic development in almost all tissues and organs of all animals and Fine-tuning of BMP is very important for its various functions.72The TGFβ family plays an important role in matrix formation and pulpal obliteration, especially in pulp-dentin pathophysiology. TGFβ induces the secretion of ECM components related to primary and tertiary dentin formation. TGFβ isoforms are also expressed by mature odontoblasts, leading to the isolation of these growth factors in the dentin matrix. This process provides a matrix-associated TGFβ library that can be released in matrix changes associated with caries injury or trauma.77The Notch signaling molecule expression and activation are critical not only for the development of tooth embryos, but also for the regeneration of damaged tissues of adult teeth. Notch is an important regulator of stem cell fate and can induce cell proliferation and differentiation. There is a close relationship between dental pulp mesenchymal cells and neovascularization in dental pulp diseases and the Notch signaling pathway is upregulated after tooth injury.82 Ectodysplasin A (EDA), a member of the TNF superfamily, and its receptor, EDAR, are an essential part of ectodermal organ development. Analysis of their expression patterns and mutant phenotypes has shown that they may participate in signal transduction between different epithelial cells during hair and tooth development in mice.82The specific role of the tumor necrosis factor (TNF) family of molecules identified in tooth morphogenesis suggests that this pathway plays an important role in the development and evolution of the tooth number and shape.Ion channels play an important role in various kinds of proprioception, pain, and conduction of hot and cold stimuli in teeth, as well as in calcium transport, deposition, and loss of teeth.88Voltage- and ligand-gated ion channels play a significant role in toothaches. The sensory fibers of the trigeminal nerve possess different types of voltage-gated ion channels expressed in common nerve cells and odontoblasts. Previous studies have shown that small molecules such as 5 ‘- adenosine triphosphate (ATP) and its ion receptor of the P2X family play an essential role in the sensory system of mediating toothaches. It is involved in the development of teeth and the formation of secondary and reactionary dentins.110 This suggests that GDNF, similar to other members of the GDNF family such as neurturin, is not involved in guiding or maintaining the neural structure during tooth development or in the survival of Schwann cells.110Although there is evidence that tooth morphogenesis and innervation are independent, the role of nerve fibers in tooth development or dentin and enamel formation remains controversial. In our study, the first molar tooth germ neurons of glial -derived neutrotrophic factor (GDNF)-deficient and wild-type mice were almost identical in structure and Schwann cell density.152Among the members of the BMP family, the biological function of BMP 2 in root development has been widely studied and it promotes the differentiation of dental pulp stem cells into odontoblasts. IGF-1 promotes osteogenic differentiation and osteogenesis of bone, but can also reduce its odontogenic differentiation and ability, suggesting that IGF-1 could be as a candidate material for bone tissue engineering. The osteogenic differentiation signaling pathway of stem cells from apical papilla (SCAP) induced by IGF-1 requires further study, whereas IGF-2 appears to preferentially play a role in enamel deposition.153Presently, the molecular regulatory mechanism of the stress response in teeth is not well understood, although it is of great significance to tooth development and the formation of secondary and reactionary dentins. The expression of heat shock protein 25 (Hsp25) in odontoblasts could be considered a stress response, which can be achieved by regulating actin dynamics and programmed cell death. Following the development of dentin and its deposition, the odontoblast retreated from the apex to the incisor end and the pulp space in the middle is reduced, forming pseudo-stratification. This histological finding suggests that odontoblasts undergo mechanical stress due to increase cell density during active dentin formation and this environmental change may induce a strong Hsp25 immune response in these cells.110 The role of nerve fibers in triggering the development of the teeth or in the onset of dentin and enamel formation is still controversial, although evidence suggests that tooth morphogenesis and innervation are independent. In our study, the first molar tooth germ (FMTG) nerves were almost identical in structure and density to the Schwann cells in GDNF-deficient and wild-type mice. This suggests that glial cell-line derived growth factor (GDNF) and neurturin are not involved in guiding or maintaining the structure of nerves in the developing tooth or in the survival of Schwann cells.110Nerve-related genes involved in the regulation of dengtinogenesis have also been reported. Some studies have shown that during pulp regeneration odontoblast cells are likely derived from Schwann cells in the pulp, but the fine regulatory mechanisms involved are still poorly understood.114 CNRs, protocadherin (Pcdh)-γ, and Reelin are related to both morphogenesis and cell differentiation events.28 E-cadherin and P-cadherin have differential and specific roles during morphogenesis115 and connexin 43 is expressed in odontoblasts.116 The expression patterns of CLDN1, OCLN, ZO-1, and ZO-2 are different. Tight junctions (TJs) of the rat lower incisor odontoblasts may play an important role in the early differentiation of odontoblasts, especially in determining the direction of mineral secretion and establishing the distal membrane domain.114The role of the cell junction in oral development and disease is poorly understood. Occludin (OCLN), claudin-1 (CLDN1), and zonula occludens-1/2 (Zo1/2) play important roles in odontoblast differentiation.117 Therefore, exploring the gene network system regulating tooth development has a very positive practical significance in the study of tooth tissue regeneration and the prevention and treatment of tooth abnormalities. The early bell stage is the initial phase of odontoblast formation and dentin matrix deposition in the tooth development process. RNA sequencing and differential gene analysis of rat tooth germ samples at the cap and early bell stages showed that the bile secretion pathway was the most significantly different between the cap and bell stages among related signaling pathway during development, which mainly included SLC10A1, SLC2A1, SLC4A4, ADCY5, ATP1B1 and ABCC3.117Recent studies have shown that this signaling pathway, ostensibly unrelated to teeth, is involved in tooth embryo development, and tooth formation involves strict genetic control procedures.118 Odontoblasts showed prominent staining for PrP at levels comparable to those of nerve fibers.122 Glucose supply via GLUT1 might occur before the differentiation of odontoblast-like cells. The transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing.144 Mdm2 promotes Odontoblast-like differentiation by Ubiquitinating Dlx3 and p53. It will continue to add and classify, because the number is small and not deep enough.However, the major signaling pathways involved in dentinogenesis and new genes remain to be discovered and elucidated. For instance, IFT140 is essential in promoting dentin formation and reparation.The development and restoration of dentin is a complicated and strict regulatory process and numerous signaling molecules and transcription factors constitute the complex signaling network mediating dentin formation. Understanding these signal networks is of great significance to the study of tooth development, damage repair, and tissue regeneration and to the treatment of abnormal tooth development. We believe that research in this field should focus on the following points in the future.155 Moreover, the old dentin structure is also different from that of young dentin, and a study on the mechanical behavior of dentin reported that all stiffness, strength, and fatigue properties tested decreased significantly with age.156 Scanning electron microscopy revealed that old dentin exhibited more intratubular crystal deposits than the young dentin, which had an 80% lower permeability.157 We hope that future research will elucidate the process and molecular mechanisms of odontoblast aging, which could provide strategies to regulate odontoblast aging or reverse the aging process.Odontoblast aging is a field that has rarely been studied in the past decade and similar to other major functional cells, odontoblasts have a process of development, maturation, and senescence. To date, the specific genes involved in odontoblast cell aging are unknown. Odontoblast aging is closely related to dentin regeneration. The autophagy-lysosome system of odontoblasts ensures the renewal of organelles and proteins, thereby extending their lifespan. However, the gradual accumulation of lipofuscin in lysosomes reduces the viability of odontoblasts and the ability of dentin to regeneration.The odontoblast is the first tissue barrier against invasion of the tooth by caries-related pathogens, and is the starting and effective point of the immune response of the tissue to these pathogens. However, the pattern recognition receptors (PRRs) involved in the responses to pathogen-associated molecular patterns (PAMPs) in odontoblasts have not been fully clarified. Therefore, a deeper understanding of the mechanisms underlying PAMP-induced innate immune responses and the role of PRRs in odontoblasts would help the development of strategies to maintain dental pulp tissues in a healthy condition for as long as possible. Furthermore, the enhanced understanding of these processes may lead to the development of novel therapeutic strategies and treatments for pulpitis.Currently, although there is considerable information available on the dentin developmental process and related genes, the details of its complex regulatory network are still unclear. Furthermore, repairing dentin abnormalities or defects that have occurred is challenging. Presently, prenatal or preimplantation genetic diagnosis is the most effective method of solving dentin developmental problems. Future further studies and understanding of the regulatory network of dentin development would contribute to enabling the modification adult mutant genes for the benefit of patients by combining modern gene modification techniques such as CRISPR/cas9.158 However, because of the limitations of in vivo and in vitro models and research techniques, epigenetic research on dentin development has only just begun. A considerable amount of research is still required to reveal rules and interventions in developmental abnormalities.Recent studies have demonstrated the important role of epigenetics in dentin development and the main epigenetic modifications include DNA methylation, histone acetylation, and methylation.159 In addition to previous studies on dentinogenesis, some novel approaches have been explored in the past year. For example, a novel injectable treated dentin hydrogel (TDMH) has been developed for use as a pulp capping material for dentin regeneration.159 Histological results showed that TDMH allowed the harvesting of thicker formed dentin than that of Biodentine and mineral trioxide aggregate (MTA).159 Another study utilized an amphiphilic synthetic polymeric combined with exosomes derived from both dental pulp stem cells and immortalized murine odontoblasts as dental pulp capping material to generate dentin in vivo. After 6 weeks, the exosome group exhibited higher quality formation of the dentin bridge than the group treated with glass-ionomer cement.154 The development of nanotechnology has also led to the application of nanomaterials in new strategies in the field of dentin regeneration. Mesoporous bioactive glass/graphene oxide composites have been shown to improved mineral differentiation of human dental pulp stem cells by upregulating the odontogenesis-specific markers DSPP and DMP-1. This observation suggests that this composite may induce stem cell differentiation into odontoblast-like cells and thereby induce dentin formation.Dentin regeneration, especially regenerative restoration after dentin injury, requires an understanding of the occurrence and development of dentin, because the two main signaling pathways are very similar. Dentin regeneration requires dental pulp cells and is closely related to dental pulp regeneration.2 spatial averaged temporal averaged intensity) treatment of teeth increased calcium ion transport-related protein  expression. After 14 days, hematoxylin and eosin (H&E) staining showed more significant dentin formation in the pulse treatment group than that in the other groups and the underlying mechanism may involve inflammatory reactions and mechanical effects.160Some physical factors including ultrasound, static magnetic field(SMF), electric field(EF), and laser irradiation also affect the differentiation of odontoblasts. Ultrasound: In the experimental model of dentin injury without pulp exposure, low-intensity pulsed ultrasound  by upregulating MMP-1 and MMP-2 gene expression. SMF also inhibits the phosphorylation of YAP/TAZ, which continuously induces odontoblast differentiation and mineralization in DPSCs.161Static magnetic field (SMF): Recently, SMF has been shown to promote the proliferation, migration, and differentiation of stem cells. Furthermore, SMF at 1 mT has been reported to increase DPSC proliferation, and the gene expression of fibroblast growth factor (FGF)-2, TGF-β, and vascular endothelial growth factor (162Electro filed (EF): A potential method  using a pulse EF to deliver growth/differentiation factor 11(GDF11) could promote DPSC differentiation into odontoblasts and induce the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts, in the future. This study suggests that the co-application of physical and gene therapy may achieve the goal of dental tissue repair.2 stimulated cellular proliferation and promoted biomineralization of stem cells from human exfoliated deciduous teeth by upregulating odontogenesis-related genes  .163 The development of genomics, molecular biology, biophysics, and materials science in the future will provide more alternative and efficient methods for the regeneration and restoration of dentin. Consequently, the “secrets” of dentinogenesis will be gradually unveiled.Laser irradiation: Low-level laser irradiation  at 8 J/cm


Here, we review the haloacid dehalogenase (HAD) class of protein phosphatases, with a particular emphasis on an unusual group of enzymes, the eyes absent (EYA) family. EYA proteins have the unique distinction of being structurally and mechanistically classified as HAD enzymes, yet, unlike other HAD phosphatases, they are protein tyrosine phosphatases (PTPs). Further, the EYA proteins are unique among the 107 classical PTPs in the human genome because they do not use a Cysteine residue as a nucleophile in the dephosphorylation reaction. We will provide an overview of HAD phosphatase structure-function, describe unique features of the EYA family and their tyrosine phosphatase activity, provide a brief summary of the known substrates and cellular functions of the EYA proteins, and speculate about the evolutionary origins of the EYA family of proteins. Among these, the HAD superfamily includes a diverse range of hydrolases including phosphoesterases, dehalogenases, phosphonatases, and sugar phosphomutases. They are characterized by the use of an Aspartate as a nucleophile in a two-step, divalent cation-dependent catalytic reaction. Evolutionarily, HAD enzymes represent one of the earliest known enzyme families. Estimates based on orthologous sequence analyses predict HAD enzymes were already present in the last common ancestor between prokaryotes, eukaryotes, and archaea [ubstrate c and staDomains inserted into the flap region or immediately after strand S3 are termed “caps” and serve to both shield the active site and provide a substrate binding cleft. Three classes of caps have been described: C0 caps are typically very small inserts, C1 caps occur in the middle of the β-hairpin flap and form a distinct structural unit , and C2 caps occur after strand S3. Some HAD family members have both C1 and C2 caps , and there are instances of C0 family members with an additional C2 cap . Cap domains serve an essential role in the catalytic activity and substrate specificity of HAD enzymes. Indeed, it has been suggested that this arrangement of an active site between a conserved catalytic core domain and diverse cap domains could contribute to the notable ability of HAD-family enzymes to explore a wide range of substrates over evolutionary time [HAD substrates vary in size and chemistry, ranging from sugars and small metabolites to proteins. There are 40 HAD phosphatase genes in the human genome with the potential to encode 161 proteins . While iAlso notable is the acquisition of a multi-domain architecture in eukaryotic HAD phosphatases, often resulting in proteins with multiple biochemical functions. For instance, soluble epoxide hydrolase 2 (sEH2) has a C-terminal epoxide hydrolase domain and an independent N-terminal HAD phosphatase domain ,4, and pUnique among the HAD phosphatases are the eyes absent proteins; they are multi-domain proteins and represent the only validated instance of tyrosine phosphatase activity housed in a HAD phosphatase domain ,5,6,7,8.2+ cofactor is poised to allow the Asp nucleophile to approach and electrostatically stabilize the substrate phosphate group. The cap domain allows for solvent exclusion. In typical HAD reactions, there is movement of the cap domain between “closed” and “open” conformations. No structural evidence for such domain movement is yet available for the EYA proteins. Furthermore, the cap domain of EYA forms a seven-helical bundle that is structurally distinct from the cap domains of all other HAD phosphatases. The helix between strands S2 and S3 that is inserted into this cap domain likely contributes to a degree of rigidity in the relative positions of the cap and core domains.The crystal structure of the catalytic C-terminal domain of EYA2 (EYA2-ED) has been determined  Figure . The EYAIn addition to being an unusual HAD family phosphatase, the EYA ED domain is also a unique tyrosine phosphatase. All classical protein tyrosine phosphatases (PTPs) are defined by the signature CxxxxxR motif that forms the phosphate binding P-loop in the active site . The nucThe structure of the EYA proteins cleanly separates individual biochemical activities by domains. The N-terminal domain houses the transactivation and putative threonine phosphatase activities, while the C-terminal domain houses the PTP activity. As a result, it is possible to link individual biochemical activities to cellular functions.Eya and Six genes are associated with severe developmental defects in humans, mice, frogs, fish, and flies, speaking to the importance of the EYA-SIX transactivation function. Among the developmental processes regulated by the EYA-SIX complex are Drosophila eye development [The cellular functions of the EYA proteins can be broadly divided into two categories: those originating from the transactivation role of EYA and those directly related to the phosphatase activities of EYA. In its transactivation role, a critical step is interaction with the SIX family of homeodomain proteins and subsequent translocation of EYA to the nucleus . Mutatioelopment , neural elopment , cochleaelopment , and limelopment . These helopment ,10,22,23Eya genes in vertebrates with some overlap in expression patterns. Furthermore, knockout strategies result in the loss of all EYA activities, making it difficult to specifically attribute a phenotype to the tyrosine phosphatase activity alone. There are two studies documenting a role for the EYA tyrosine phosphatase activity in normal development. We have shown that pharmacological inhibition of the EYA PTP activity delays vascular development in the post-natal mouse retina [Drosophila Eya proteins was initially implicated in eye formation using GAL4-UAS mediated genetic rescue experiments [Drosophila appears to be that, while the Eya PTP activity contributes to development, it is not essential [sine oculis or dachshund) compromised the ability of PTP-dead Eya to rescue retinal defects [Elucidation of the contribution of the EYA tyrosine phosphatase activity to normal development is complicated by the fact that there are four e retina . Curiouseriments ,25, subseriments . The emessential . A recen defects . In addi defects .The best understood cellular process in which EYA PTP activity plays a role is DNA damage repair. In eukaryotic cells, double strand break repair is initiated by a cascade of signaling events leading to the formation of a DNA damage repair complex a. One ofOther proposed substrates of the EYA PTP activity include estrogen receptor beta ; a docking surface(s) distinct from the active site engages the substrate and confers specificity . FurtherPyromices finnis, an obligate anaerobic chytrid fungus that grows in the gut of mammalian herbivores and is necessary for digestion of plant cell walls. Motifs I–III of the HAD phosphatases are present in the fungal ED, and motif IV is present in four out of five fungal EYAs (Monosiga brevicolis [EYA orthologues have been reported across the animal kingdom , as wellgal EYAs b. A quergal EYAs c. Indeedevicolis , commonlevicolis . We idenevicolis , suggestTaken together, this examination of the origins of the EYA domain suggests that a proto-EYA likely diverged and evolved independently in each of the downstream metazoan kingdoms. The observations described above are also compatible with the EYA domain evolving independently of the other HAD phosphatases. In the absence of experimental characterization of early putative EYA orthologues, it remains a matter of speculation whether the early EYA domain containing proteins had tyrosine phosphatase activity. Pertinent to this discussion, it has been proposed that classical PTPs evolved before protein tyrosine kinases, and that this was driven by the ability of some promiscuous serine/threonine kinases to also phosphorylate tyrosine residues .There is an expanding recognition that the eyes absent proteins play important roles in normal development and are important contributors to disease pathologies ,10. They


Prior research has documented the health and well-being effects of volunteerism in later life, and that positive outcomes increase in the first 100 volunteer hours/year and slightly increase between 100-200 hours. Given this, it seems that using an intersectional lens to explore disparities in volunteer behaviors and what might explain them is important from a health equity standpoint. Using data from 268,194 individuals aged 65-85 from the most recently available Volunteer Supplement of the Current Population Survey, this study found that White older adults were most likely to spend any time in volunteer activities, while Asian and Hispanic older adults were least, across all racial/ethnic groups. Further, the percentage of older Asian women volunteering in the 100-200 hour range (27%) was significantly higher than that of older Asian men (19%). Social and cultural factors that may explain these racial/gender differences and implications for recruiting older adults as volunteers are discussed.


Human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as those of the oropharynx. HPV proteins activate host DNA damage repair factors to promote their viral life cycle in stratified epithelia. Activation of both the ATR pathway and the ATM pathway are essential for viral replication and differentiation-dependent genome amplification. These pathways are also important for maintaining host genomic integrity and their dysregulation or mutation is often seen in human cancers. The APOBEC3 family of cytidine deaminases are innate immune factors that are increased in HPV positive cells leading to the accumulation of TpC mutations in cellular DNAs that contribute to malignant progression. The activation of DNA damage repair factors may corelate with expression of APOBEC3 in HPV positive cells. These pathways may actively drive tumor development implicating/suggesting DNA damage repair factors and APOBEC3 as possible therapeutic targets. Human papillomaviruses (HPV) are small, nonenveloped viruses that contain double-stranded DNA genomes of approximately 8 kb in length. More than 400 different genotypes of HPV have been identified, and each infects stratified epithelial tissues at distinct body locations . Over onHR-HPV genomes encode an average of eight open reading frames (ORFs) that are expressed from polycistronic messages that are transcribed from one strand of viral DNA . The indThe productive life cycle of HPV is closely linked to the differentiation state of the host epithelial cell and is regulated through the action of both viral as well as cellular proteins ,21,22,23The DNA damage response consists of a network of cellular signaling pathways that facilitate the repair of damaged cellular DNA to maintain genomic integrity. This includes mechanisms to repair damaged DNA and, in some cases, induce apoptosis or senescence, as well as activation of an innate immune response . The twoThe ATR pathway is activated in response to replication stress and the presence of single-stranded DNA (ssDNA) at stalled replication forks. ssDNA is bound by the replication protein A (RPA), which recruits ATR and its binding partner, ATRIP, to sites of damage . The recActivation of the ATM and ATR DNA damage repair pathways is critical for the completion of the differentiation-dependent HPV life cycle ,37,38. BIn addition to its direct effects on DNA damage repair, ATR also indirectly regulates expression of a set of cellular genes. Activation of the ATR pathway in HPV positive cells is the result of complex formation of ATRIP and TOPBP1 which are all recruited to viral replication centers . TOPBP1 The mechanisms responsible for constitutive activation of DDR pathways in HPV positive cells are beginning to be understood. Cells that stably maintain HPV episomes or those that express only E6 or E7 exhibit significantly enhanced levels of DNA breaks in comparison to normal cells ,47. In HInfections by high-risk HPVs in the genital tract produce asymptomatic lesions or dysplasias that are graded according to morphologic changes observed in the differentiation pattern. Cervical intraepithelial neoplasia (CIN)1 is characterized by koilocytic changes whereas CIN2 and CIN3 define moderate and severe dysplasia, respectively. Importantly, examination of biopsy materials revealed that the distribution and levels of DNA damage repair (DDR) factors increase with the grade of cervical lesion . This suThe expression of both high-risk E6 and E7 is necessary for the immortalization of cells. The high-risk E7 proteins are localized to the nucleus where they bind the retinoblastoma, Rb, proteins and induce their rapid degradation. In normal cells, Rb proteins bind inhibit the action of E2F transcription factors that regulate expression of genes involved in replication in the S-phase. As cells transit into the S-phase, cyclin-kinase complexes phosphorylate Rb leading to release of E2F factors and activation of expression of replication genes. By inducing the degradation of Rb, E7 constitutively activates E2F, which leads to rapid entry into the S-phase . The actHPV genomes are maintained as extrachromosomal episomes in precancerous lesions; however, they are frequently found integrated into host chromosomes in cancers . Viral iTRAF3) and ATM genes [E2F1 gene, is found in approximately 20% of HPV positive OPSCC while in only 2% of HPV negative OPSCC [Despite high level activation of DNA damage repair pathways, HPV positive lesions accumulate large numbers of mutations that contribute to progression to cancers. Somatic mutations have been recognized as a key mechanism of carcinogenesis by inducing driver mutations in a number of genes. Whole exome sequencing analyses of cervical cancer biopsy materials have identified recurrent somatic mutations in genes such as EP300 FBXW7, PIK3CA, TP53, MAPK, PTEN, ERBB2 and STK11 . InteresTM genes . In addive OPSCC . Althougve OPSCC .APOBECs are a family of cytidine deaminases that function to convert cytosine to uracil in ssDNA. At least 11 different types of deaminase are present in humans and contain either a single  or double  cytidine deaminase domain ,68,69. IPersistent infection by HPV results in APOBEC induced mutations in cellular DNAs that can contribute to malignant progression . HR-HPV Several studies have suggested a linkage between DDR pathways and APOBECs. The repair of DNA breaks by the ATR pathway often generates ssDNAs which can be targeted by APOBECs ,86,87. TRecent studies have suggested that DDR inhibitors such as ATR and Chk1 inhibitors may sensitize tumor cells with high levels of APOBEC to cell death ,91,92. FHigh risk HPV proteins manipulate cellular pathways to promote their viral life cycles. HPVs constitutively activate the ATM and ATR DNA repair pathways and preferentially recruit these factors to viral genomes for productive viral amplification. Persistent infection by HPV results in increased levels of APOBEC3 proteins that result in TpC mutations in cellular DNAs that contribute to malignant progression. Targeting of APOBEC and/or DDR factors in HPV related cancers may be effective therapeutic targets.