Investigate the prognostic value of the fat necrosis deposit (FND) pattern in acute pancreatitis.The contrast-enhanced computed tomography (CT) images of 35 necrotizing pancreatitis (NP) and 51 edematous pancreatitis (EP) cases were included in our retrospective study. Computed tomography severity index (CTSI) and Ranson scores were calculated. Images were evaluated for FND, complications (infection/ hemorrhage), walled-off necrosis (WON), and venous thrombosis (VT). We developed a new grading system called fat necrosis deposit-CTSI (FND-CTSI), which was the sum of FND and CTSI scores. The relationship between grading systems and mortality, length of hospital-intensive care unit stay, surgical and percutaneous interventions were evaluated.FND-CTSI scores were significantly higher in NP than EP (P < 0.001). FND-CTSI demonstrated a significant correlation with CTSI  and Ranson score . CTSI was significantly higher in only mass form amongst the FND groups (P < 0.001). There was a significant difference in WON, complications, and mortality between FND groups (P < 0.05). CTSI and FND-CTSI scores were both significantly associated with WON, VT, surgical intervention, mortality (P < 0.001), and the presence of complications . FND-CTSI was also significantly associated with percutaneous intervention (P = 0.019), while CTSI was not (P > 0.05). According to ROC analysis, AUC values of FND-CTSI were higher than CTSI for the detection of WON, complications, mortality, and percutaneous intervention (P < 0.05). FND-CTSI showed a highly significant correlation with the length of hospital and intensive care unit stays (P < 0.001). FND-CTSI can be used in acute pancreatitis grading and considered as a prognostic factor.  The prevalence of acute pancreatitis is estimated to be 10–50/10,000 and the mortality rate is approximately 6% [1–3]. Fatty tissue, fascia, and adjacent organs may also be affected in addition to the pancreas parenchyma in acute pancreatitis. Ranson [4], acute physiology and chronic health evaluation (APACHE II) [5], and CT severity index (CTSI) have been used as a clinical scoring system for the prognosis prediction of patients with acute pancreatitis for a long time .Pancreatic necrosis generally appears homogeneous initially and later become heterogeneous by liquefication [8]. The severity of necrotizing pancreatitis (NP) depends on the necrosis ratios and is classified as <30%, 30%–50%, and >50%. Follow-up imaging is advised for patients with less than 30% necrosis which can resemble interstitial edematous pancreatitis [6]. Fat necrosis is a well-known complication of acute pancreatitis [9]. The most common localizations are peripancreatic fat tissue, omentum, and mesenteric fatty tissue [10]. Isolated peripancreatic necrosis is observed in less than 20% of cases, and these patients have a better prognosis than the ones with parenchymal necrosis [11]. Peripancreatic necrosis is considered when increased attenuation, linear stranding, and fluid collection is observed in peripancreatic fatty tissue. However, these findings can also be observed in acute interstitial edematous pancreatitis (EP). Heterogeneity in high attenuation suggests peripancreatic necrosis. A combination of both parenchymal necrosis and peripancreatic fat necrosis is the most common form of NP with a prevalence ranging between 75%–80% [8]. Fat necrosis can present as a peritoneal nodule, mesenteric implant, or mass in acute pancreatitis [12–14]. These findings may resemble peritoneal carcinomatosis or primary peritoneal cancer [14]. Therefore, it is suggested to evaluate patients with clinical findings and older examinations to avoid misinterpretation of these peritoneal and mesenteric lesions [15].We observed fat necrosis deposit (FND) in patients who had severe pancreatitis in our daily practice. However, the FND pattern was different amongst the patients. Therefore, we aimed to investigate the prognostic value of the FND pattern in acute pancreatitis. Acute pancreatitis was diagnosed according to the Revised Atlanta Classification System [16] in our study. A total of 86 patients were included, of which 35 (40.7%) were NP and 51 (59.3%) were EP. Informed consent was waived in our retrospective, and the institutional review board approved the study. Images were performed on 128-sliced CT  scanner. Imaging parameters were as follows: tube voltage = 130 kV, effective mAs = 90, slice thickness 1mm, collimation = 2 × 4 mm, pitch = 1.6. Images were obtained at 70 s after intravenous administration of 100 mL Iopromide  at a speed of 3 mL/s. Images were evaluated with Picture Archiving and Communication System (PACS), OsiriX MA v 10.0.1 , GPL licensed free access resource code, and commercially licensed, FDA approved Mac OS X radiology work station.The abdominal CT images of the patients with acute pancreatitis performed 2–5 weeks after the disease onset were recruited because the liquefied components become more apparent after 1 week . The images were evaluated independently by two radiologists Ş.E and M.Ç, who have 10 and 3 years of abdominal CT imaging experience, respectively. Each patient was evaluated according to the CTSI (Table 1) [17] and Ranson scoring system [4]. Images were also evaluated for complications , pseudocyst, walled-off necrosis (WON), and venous thrombosis (VT) . Revised Atlanta Classification System classifies NP associated collections according to the disease onset time. Collections within 4 weeks without a wall are defined as the acute necrotic collection, whereas collections persisting after 4 weeks with an encapsulated wall are defined as WON . The presence of Diabetes Mellitus (DM) was searched via electronic medical records. The scoring system according to the FND pattern was scored as follows; none:0, only peripancreatic:2 , anterior pararenal/mesenteric:4 , beyond Gerota’s fascia:6 , and mass form:8 . Pancreatic necrosis in CTSI and pancreatic-peripancreatic necrosis in Modified CTSI are defined by ordered even numbers [7]. Therefore, we used ordered even numbers in our FND scoring system which also enables statistical calculations. In addition, we developed a new grading system called Fat Necrosis- CT Severity Index (FND-CTSI) as the sum of FND and CTSI scores. Mortality, duration of hospital stay , duration in the intensive care unit , surgical , and percutaneous interventions were evaluated as prognostic factors. Statistical analyses of the study were performed by SPSS  with version 20.0. Power analysis was performed by GPower software . The number of patients required for the study was determined by power analysis for Chi-square analysis of NP and EP groups with 0.30 effect size, 5% error, and 80% power. Groups were determined by the single blinding method. Descriptive statistics were presented as frequencies (percentage) for categorical variables; as median and interquartile range (IQR) for numerical variables. Continuous variables were analyzed for normality by the Kolmogorov-Smirnov test. The associations between variables amongst categorical data were determined by the corrected Chi-square analysis. Since the distributions of the measurements were not normal, the Mann-Whitney U test for two independent samples and the Kruskal-Wallis test for multiple samples were used for group comparison. Next, pairwise comparisons with Bonferroni correction for multiple testing were performed. Receiver Operating Characteristic (ROC) curve analysis was done to examine the performance of CTSI and FND-CTSI using Area Under the Curve (AUC). A p-value of less than 0.05 (P < 0.05) was considered statistically significant by taking 5% for type-I error.The mean age was 51.78±13.85 and 56.74±16.63 in the NP and EP group, respectively. The percentage of male and female patients were 52.3 and 47.7, respectively. There was no significant relationship in gender distribution and mean age between NP and EP groups . Pseudocyst was only observed in EP (9/51) (17.7%), WON was only observed in the NP group (29/35) (82.8%). WON was observed in the intraparenchymal and peripancreatic distribution in 86.5%, intraparenchymal in 8.1%, and peripancreatic in 5.4%. VT was observed only in the NP group (9/35) (25.7%) (P < 0.001). There was no significant difference in the presence of complications (P = 0.07) and DM (P = 0.69) between the groups. Inter-reader reliability for FND-CTSI was calculated as excellent . FND distribution was as follows; none (n=60), only peripancreatic (n=6), anterior pararenal/mesenteric (n=12), beyond Gerota’s fascia (n=3), and mass form (n=5). The ratio of the presence of FND in the NP group was 26/35 (74.29%). CTSI was significantly higher in only mass form FND group (P < 0.001), but there was no significant difference between the other groups (P > 0.05). FND-CTSI scores exhibited a compatible increase with FND distribution (P < 0.001). There was a significant difference in WON (P = 0.05), complications (P = 0.004), and mortality (P = 0.006) between the FND groups. The rate of complications and mortality was observed more in the mass form group, while WON was more in the beyond Gerota’s fascia group. .There was no significant difference in the Ranson score  between the groups (P = 0.18).FND-CTSI demonstrated a significant and very strong correlation with CTSI . FND-CTSI demonstrated a significant and weak correlation with Ranson . CTSI and FND-CTSI were significantly higher in NP  group than the EP group  (P < 0.001)FND-CTSI was significantly associated with necrosis ratio (P < 0.001). CTSI and FND-CTSI scores were both significantly associated with WON, VT, surgical intervention, and mortality (P < 0.001). CTSI and FND-CTSI scores were also significantly associated with the presence of complications . FND-CTSI was significantly associated with percutaneous intervention (P = 0.019), while CTSI was not (P > 0.05). None of the scores were significantly associated with DM (P > 0.05) (Table 2). According to ROC analysis, AUC values of CTSI and FND-CTSI for WON, VT, complication, mortality, and surgical intervention were statistically significant (P < 0.05), but not for DM. FND-CTSI showed higher AUC for the detection of WON, complication, mortality, and percutaneous intervention whereas CTSI showed higher AUC for the detection of VT and surgical intervention (Table 3). According to cox regression analysis, both scoring systems showed a highly significant and positive correlation with the length of hospital  and intensive care unit  stays. CTSI and FND-CTSI scores were both significantly higher in the groups with longer duration of hospital stay and duration in the intensive care unit (P < 0.001) (Table 4).FND-CTSI is a newly developed CT grading, which is composed of CTSI and FND scores. To the best of our knowledge, this is the first study to investigate the value of FND in CT grading of acute pancreatitis. There was a significant difference in WON, complications, and mortality between FND groups. FND-CTSI was significantly associated with necrosis ratio, WON, VT, complication, mortality, and a need for surgical or percutaneous intervention. It also showed a highly significant and positive correlation with the length of hospital and intensive care unit stays.Intraabdominal fat is located in both of the retroperitoneal and intraperitoneal compartments . Fat necrosis is a known complication of acute pancreatitis [9]. The most common localizations are peripancreatic fat tissue, omentum, transverse mesocolon, and mesentery . Although retroperitoneum is a common localization for pancreatitis and fat necrosis, it is generally limited within anterior and posterior pararenal spaces. It is suggested that fat necrosis is caused by the discharge of lipase into the lymphatic and vascular system during acute pancreatitis [10]. Fat necrosis occurs as a result of fat saponification by released lipolytic enzymes from the affected parenchyma . The affected fat tissue activates the macrophage and other inflammatory mediators and aggravates the inflammatory response . Phospholipase and protease attack the plasma membrane of fat cells, which release triglycerides. Free fatty acids are formed by hydrolysis. Then, calcium soaps are formed by the combination of fatty acids and calcification. This theory is suggested as the reason for hypocalcemia in severe pancreatitis. Fat necrosis deposits are distributed in the retroperitoneum and abdominal cavity after the resolution of acute exudate and ascites [23]. These nodules may cause mass effect and late enhancement because of the slow diffusion of contrast material from the capillaries in granulation tissue . The extrapancreatic spread of exudate is a common complication of pancreatitis. It is generally observed in retroperitoneum and anterior pararenal spaces but doesn’t spread beyond renal fascia. Thus, kidneys and perirenal fat tissue are not affected [19]. On the other hand, there are cases of subcapsular and intrarenal pancreatic fluid collections in the literature [25]. Gerota’s fascia is thought to act as a barrier between pararenal and perirenal spaces [24]. In our study, most of the NP cases had FND and the majority of them were distributed in the anterior pararenal space. However, we also observed patients with FND distributed beyond Gerota’s fascia. In our study, there were also some patients whose FND was in mass form. Fat necrosis can also present as a gross palpable anterior abdominal mass [12]. Pedrosa et al. presented a case of a renal pseudotumor caused by retroperitoneal fat necrosis secondary to acute pancreatitis. Fat necrosis should be kept in mind for the differential diagnosis of renal tumors in patients with acute pancreatitis. Progressive and prolonged contrast enhancement can help to identify the pseudotumor in such cases [13]. Smith et al. described fluid collections, solid enhancing peritoneal nodules, mesenteric implants, and bulky soft tissue lesion surrounding vascular structures at the mesenteric root. They interpreted these findings as a result of exuberant granulomatous reactions secondary to acute pancreatitis. The findings resembled peritoneal carcinomatosis or primary peritoneal carcinoma [14]. Therefore, it is necessary to differentiate nodular fat necrosis from peritoneal malignancies by evaluating clinical-laboratory findings and previous imaging findings showing pancreatitis [15]. The CT findings of some of the patients in our study were also seemed to be a mimicker of peritoneal carcinomatosis.Inflammation, pancreatic necrosis, and local complications are the evaluated factors to differentiate between mild acute pancreatitis  and severe pancreatitis (necrotizing). This enables to implement the precise treatment management . In our study, both CTSI and newly developed FND-CTSI scores were higher in NP than EP. FND-CTSI demonstrated a significant correlation with both CTSI and Ranson scores. CTSI was significantly higher in only mass form amongst the FND groups.Extrapancreatic complications such as pleural effusion, ascites, vascular complication , parenchymal complication , and gastrointestinal involvement  were evaluated in addition to the pancreatic findings to determine the severity of acute pancreatitis in the previous studies . However, none of these studies evaluated the FND pattern. An increase in the FND score resulted in a significant increase in complications and mortality. We observed that both FND-CTSI and CTSI were significantly higher in patients who needed surgical intervention, similar to the studies in the literature. Both scores were also significantly associated with WON, VT, complication, and mortality. FND-CTSI was also significantly associated with percutaneous intervention, while CTSI was not. Balthazar et al. observed an excellent correlation between CTSI and necrosis, duration of hospital stay, complication development, and death . CTSI and pancreatic necrosis were significantly associated with the need for intervention [29]. Leung et al. declared that CTSI was more sensitive than the Ranson score in the prediction of complication, mortality, and duration of hospital stay [30]. FND-CTSI provided better diagnostic performance for the detection of WON, complication, mortality, and percutaneous intervention, whereas CTSI showed better diagnostic performance for the detection of VT and surgical intervention in the current study. CTSI and FND-CTSI had a significant association and correlation with the length of hospital and intensive care unit stay in our study similarly with the literature.The limitations of our study are as follows: limitations adherent to the retrospective design; we could only evaluate the prognosis of the patients that we could manage the information from the electronic medical records, small patient population.Acute pancreatitis can cause fat necrosis. Fat necrosis deposits are primarily seen in necrotizing pancreatitis. FND-CTSI is associated with the severity of disease and prognosis. Therefore, we suggest that FND-CTSI can be used in acute pancreatitis grading, and the grade of FND pattern may be a prognostic factor that can help with clinical decision making regarding the treatment.


Androgen receptor signaling pathway is able to regulate biological processes in Sertoli cells as well as germ cells surrounded between Sertoli cells. Our review will summarize the major discoveries of androgen receptor signaling pathway in Sertoli cells and the paracrine action on germ cells. Androgen receptor signaling pathway regulates Sertoli cell proliferation and maturation, as well as maintain the integrity of blood-testis barrier formed between Sertoli cells. Also, Spermatogonia stem cells achieve a balance between self-renewal and differentiation under androgen receptor signaling regulation. Meiotic and post-meiotic processes including Sertoli cell - Spermatid attachment and Spermatid development are guaranteed by androgen receptor signaling until the final sperm release. This review also includes one disease related to androgen receptor signaling dysfunction named as androgen insensitivity syndrome. As a step further ahead, this review may be conducive to develop therapies which can cure impaired androgen receptor signaling in Sertoli cells.Androgen receptor signaling pathway is necessary to complete spermatogenesis in testes. Difference between androgen binding location in Sertoli cell classifies androgen receptor signaling pathway into classical signaling pathway and non-classical signaling pathway. As the only somatic cell type in seminiferous tubule, Sertoli cells are under androgen receptor signaling pathway regulation  Male infertility is currently a major problem worldwide and causes substantial psychological and social distress . It not Spermatogenesis is a complex process that is under precise regulation. Starting from spermatogonia stem cell (SSC) producing differentiated spermatogonia, spermatogonia transform into spermatocytes which then undergo meiotic divisions to produce round spermatids . Round sAs the only somatic cell type in seminiferous tubules , SertoliWe surveyed articles in the PubMed database using the following search terms: androgen receptor*, Sertoli cell*, spermatogonia*, maturation and differentiation*, spermatogenesis*, meiosis*, spermatid* and androgen-insensitive syndrome*. We will present this review at the cellular/molecular level in six parts: SC proliferation and maturation, SSC self-renewal and differentiation, spermatocyte meiosis, BTB integrity maintenance, Sertoli cell - Spermatid adhesion, and sperm release Figure 1The gene encoding AR is located on the X chromosome, belonging to nuclear receptor subfamily 3, group C, member 4. It consists of 8 exons that encode 4 domains: an N-terminal domain (exon 1), a DNA-binding domain (exon 2 and exon 3), a hinge region that contains the nuclear localization signal (exon 3 and exon 4) and a ligand-binding domain (exons 4-8) , 18.The N-terminal domain contains activation function domain-1 which is constitutively active . The DNAIn adult rodent testes, the AR expression level in SCs is low except during stage VI-VIII of the seminiferous epithelium . In men,Stimulated by luteinizing hormone in the pituitary gland which is part of the hypothalamus-pituitary-gonadal axis, Leydig cells produce androgen. When androgen saturates the AR of SCs and binds to AR, the AR signaling pathway is activated . Testostvia its ligand-binding domain  HSP23, HSP40, HSP56, HSP70, HSP90 g domain . When ang domain , 36. Witg domain , 38. In g domain . Howeverg domain , 40. Notg domain .Rsk). Activated p90Rsk translocate into nucleus, where it activates transcription factors such as cAMP-response element-binding protein (CREB), which binds to the cAMP-response element (CRE) of the targeted gene to regulate transcription , testosterone binds to the AR located in the cell membrane and activates the non-classical AR signaling pathway within 1 minute . In the cription , 43.in vitro study in which the TM4 cell line was exposed to testosterone levels of 10-100 nM demonstrated that testosterone can also activate a second non-classical signaling pathway, the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, directly by activating the PI3K subunit p85α. Phosphorylated Akt can activate Src and this facilitates the translocation of cytoplasmic AR to the plasma membrane , a novel membrane-bound androgen receptor unrelated to classic AR, was found in the 93RS2 Sertoli cell line which has no cytoplasmic AR. ZIP9 participates in the phosphorylation of CREB and ATF1 e future .SC proliferation occurs during fetal and early neonatal life in rodents, and in the fetal and peripubertal periods in higher primates when SCs undergo mitosis to increase their number . During Tfm) model that lacks functional AR in all cells  in mice and rats through classical signaling pathway  finger protein 4 (RNF4) binds to AR and promotes porcine SCs proliferation by elevating Proliferation cell nuclear antigen (a marker of cell proliferation) mRNA transcript levels  have been found to regulate SC proliferation and maturation. miR-124a binds to the 3’-UTR of t levels . miR-762t levels . Moreovet levels . Additiot levels  and miR-t levels , which rt levels Figure 2Ube2b) which regulates the expression of genes related to glycosylphosphatidylinositol (GPI)-anchor biosynthesis and oxidative phosphorylation , a key transcription suppressor gene, has been characterized as a marker for undifferentiated SSCs in rodents  to inhibit its expression; this can further decrease WT1 transcription factor (Wt1) expression since Wt1 expression requires binding of GATA-2to its promoter. Wt1 binds to β1-integrin and increases its expression in SCs; β1-integrin will interact with unknown molecules on the surface of SSCs, to induce Plzf expression. As a result, activation of classical AR signaling pathway can promote SSC differentiation by downregulating Plzf expression. We propose that the exact molecules that interact with β1-integrin on SSC surface should be identified to determine a more concise relationship between SCs and SSCs (Promyelocytic leukemia zinc finger ( rodents  and prim rodents . Plzf isntenance . Recentland SSCs .Igf3) mRNA transcript level was found to be upregulated by androgen; this can promote SSC differentiation through non-classical signaling pathway (Rhox5), which is directly regulated by classical AR pathway, also contributes to spermatogonia differentiation  secretion by Sertoli cell  models  models . Moreove) models . These r) models Figure 3) models . Althoug) models . PreciseWe conclude that there are two causes of meiotic arrest: chromosomal dysfunction and germ cell apoptosis.Egf, betacellulin (Btc) and neuregulin 1 (Nrg1). These EGFs bind to their corresponding receptors  on the surface of spermatocytes and stimulate the accumulation of above homologous recombination factors for appropriate prophase preparation  during stages VII-VIII of spermatogenesis when BTB undergoes restructuring  targeting TJ proteins (ZO-1 and occludin) and basal ES proteins   .Based on the results described above, we hypothesize that although the transition of preleptotene spermatocytes into adluminal compartment is not blocked when AR signaling in SC is prevented, the amount of claudin available for remodeling BTB  is insufficient under these conditions. Besides, the downregulated tight junction proteins are not sufficient to enter the nucleus and increase their own transcription, let alone to recruit more claudins to the BTB. While Occludin and Claudin26 show relatively upregulated mRNA transcript levels, this is not enough to compensate for the loss of other tight junction proteins. Thus, humoral immunity against self-antigens is mounted, resulting in disruption of normal spermatogenesis.via non-classical signaling pathway in vivo (Connexin 43 (Cx43), one gap junction protein, also under AR regulation as shown by its reduced mRNA transcript levels and protein expression levels in adult pig testes treated with flutamide, an anti-androgen . Interes in vivo . The rel in vivo .via clathrin- and caveolin-mediated pathways . The proteins that form ES disassemble before sperm release during stage VIII. The complexes connecting SC and elongated spermatids are tubulobulbar complex and the focal adhesion-related disengagement complex. AR signaling pathway is important in two processes during spermiogenesis and final spermiation: Sertoli cell – Spermatid adhesion and sperm release 3.in vivo model. First, in rats that received subdermal testosterone and oestradiol (TE) implants to lower the intratesticular testosterone level, researchers found loss of stage VIII and late spermatids  is significant in this process, mainly by participating on the nuclear export and transport of specific mRNAs as well as the structural integrity of Chromatoid Bodies of round spermatids . As an RPicalm) and early endosome antigen 1 (Eea1) and thereby inhibit their expression. Coactivator steroid receptor coactivator-1 (Src1) can be recruited to ARE of syntaxin 5 (Stx5a) and induce its expression  to the ARE of phosphatidylinositol binding clathrin assembly protein  is critical for sperm motility  is an X-linked recessive disorder of sex development in patients with the 46, XY karyotype . AccordiCurrently, researches on AIS focus mainly on recording patient phenotypes and detecting AR mutations through sequencing of AR gene. Various AR mutations have been found to date . Inadequvia aromatase and luteinizing hormone can stimulate testicular estrogen synthesis. Researchers have found high levels of luteinizing hormone and testosterone in AIS patients  an undescended testis has an increased risk of malignant transformation after puberty; and 2) testosterone produced by the testis can be converted to estrogen, so puberty will occur naturally , 186. BeMoreover, the job of a doctor is not only to treat patients physically, but also to treat them psychologically. Helping AIS patients overcome their psychological disorders and live a normal life without discrimination is a responsible behavior for doctors and for society as a whole.Both classical and non-classical androgen receptor signaling pathway are essential for spermatogenesis. These two pathways not only regulate genomic action in Sertoli cells but also influence germ cells between Sertoli cells through paracrine action. Sertoli cells are expected to be a new therapeutic target. However, rare studies focus on the relationship between androgen receptor signaling pathway, Sertoli cell and clinical diseases. The transgenic model developed recently which is able to activate only classical or non-classical signaling pathway, allows us to screen more genes related to androgen receptor signaling pathway. These genes are helpful for us to develop therapies for treating infertility. Moreover, spermatogonia transplantation is a method to inject spermatogonia into seminiferous tubule lumen and promote spermatogonia to form colonization at basement area, which may cure fertility fundamentally. The problem about making spermatogonia migrating from adluminal area to basement area may be solved by androgen therapies because lowering testicular testosterone level can open BTB reversibly.J-MW, Z-FL, and W-XY conceived of and authored the paper. All authors contributed to the article and approved the submitted version.This work was supported in part by National Natural Science Foundation of China (No 32072954).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling—the preferential activation of a signaling transducer in detriment of another—have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling. CB1 was first described in rat , lea, leai/o  release . Althougg to Gαs , about 1CP55,940 . As a remulation ,94,95. Ureceptor , but WINresponse . CB1 Gα1uggested  due to Ated  via the scaffolding of mitogen-activated protein kinases GTPγS binding induced by CP55,940 in cortex membranes, indicating a loss of G-protein desensitization [35S]GTPγS binding in hippocampus and cortex, while [35S]GTPγS binding in the cerebellum was unchanged GTPγS binding in the PAG and spinal cord was reduced by chronic THC treatment in WT but not in β-arrestin2 KO mice, while no changes were found in the preoptic area of the hypothalamus for either genotype. These findings indicate that β-arrestin2 regulates desensitization of CB1 induced G-protein activity in PAG, spinal cord, and preoptic area of the hypothalamus, and that desensitization by β-arrestin2 is the underlying mechanism behind the development of tolerance to cannabinoid antinociception and hypothermia. Interestingly, the development of tolerance to THC catalepsy was enhanced in β-arrestin2 KO mice, and agonist-stimulated [35S]GTPγS binding was reduced in basal nuclei—globus pallidus and substantia nigra—after chronic THC treatment in β-arrestin2 KO but not in WT mice GTPγS binding, for instance. In another study, Laprairie et al. [i/o-dependent pERK1/2 response and β-arrestin1 recruitment, they found that THC and CP55940 show β-arrestin1 biased signaling, while the endocannabinoids 2-AG and AEA show Gαi/o biased signaling. Since pERK was used to assess Gαi/o signaling, it is necessary to exercise caution when analyzing these data, as pERK1/2 is a response that can be elicited by other G-proteins as well as by β-arrestins, which may be a confounding factor. In addition, β-arrestin2 recruitment was not investigated since STHdh cells do not express β-arrestin2. Nonetheless, CP55940 and THC were detrimental to cell viability, while 2-AG, AEA, and WIN55212-2 improved viability in cells expressing mHTT. This finding suggests that CB1 G-protein signaling is neuroprotective in HD.CB1 ligands are capable of functional selectivity, but clear biased-signaling profiles have been challenging to characterize reliably across different studies. Laprairie et al.  comparede et al.  investige et al.  with WINIn a different study, Khajehali et al.  investigMore recently, Zhu et al.  evaluateIn contrast, Ibsen et al.  investigOrthosteric agonists, antagonists, and inverse agonists bind to the primary, orthosteric, binding pocket and compete for binding with endogenous ligands. On the other hand, allosteric ligands bind to an allosteric site, which is topologically distinct from the orthosteric binding pocket and do not compete for binding with orthosteric/endogenous ligands . A liganH-indole-2-carboxamide analog , a 1e analog A that waRemarkably, ORG enhanced CP55940-mediated pERK1/2 signaling, and also stimulated this signaling pathway when administered alone ,126,127.Studies on the impact of ORG on receptor conformation have begun to shed light on the molecular mechanism for β-arrestin recruitment and signaling at the CB1 receptor. Using site-directed fluorescence labeling, Fay and Farrens  showed tWhen administered in vivo, ORG had no effect on CP55940-mediated antinociception or catalepsy ,134. Int1.49 and Arg4097.65. This was confirmed by mutational analysis, as in an E133G CB1 mutant, pregnenolone has no effect on THC-mediated suppression of cellular respiration [An endogenous NAM for CB1 has also been described. Pregnenolone, 3α-hydroxy-5β-pregnan-20-one B, is a spiration . Bindingpiration ,132. TheCannabis intoxication and to reduce Cannabis abuse potential. Importantly, pregnenolone was also shown to block a cannabinoid-induced psychotic-like state in mice [Cannabis become more popular, pregnenolone or potential novel analogs could become an important tool in the clinic. However, its efficacy for inhibiting signaling from endogenous cannabinoids has not yet been investigated and could be a source of adverse effects.In rodents, pregnenolone prevented THC-induced increases in food intake and memory impairment. Further, neuronal firing in the ventral tegmental area and dopamine release in the nucleus accumbens induced by THC were reduced by pregnenolone. Accordingly, pregnenolone also reduced WIN55212-2 self-administration . These f in mice . As mediGAT211 is a compound derived from 2-phenylindole C that haThe therapeutic potential of GAT211 and its enantiomers has been shown in several preclinical models. GAT211 and enantiomers enhanced cell viability in a striatal cell line expressing mHTT, and improved motor coordination and prevented motor impairment in the R6/2 mouse model of HD . In a prHowever, GAT211 is a probe compound not intended to be developed for the clinic, due to its low affinity for CB1 and rapid metabolic clearance. To address these issues, fluorinated analogs of GAT211 were developed—GAT591 and GAT593 C. These i/o proteins. However, signaling promiscuity is reported throughout the literature. In addition to Gi/o, CB1 has been shown to couple to Gs [q/11 [12/13 [i/o protein activation is reportedly small, it is possible that activation of different G-protein subtypes is associated with different CB1 functions. For instance, while Gi/o is responsible for the suppression of synaptic neurotransmitter release [12/13 activation may be responsible for CB1 mediated regulation of neurite growth [i/o signaling affects cannabinoid-induced physiological effects must be studied further to ascertain whether shifting G-protein subtype preference could pose a therapeutic advantage when targeting CB1. Regardless of which G-protein is coupled by CB1, the mechanism of activation culminates in an outward movement of the intracellular domain of TMH6, while the G-protein subtype flexibility is likely due to weak interactions of the receptor ICL2 with the Gα on the intracellular surface [i/o proteins, which could elucidate the role of these signaling pathways on cannabinoid function. This activation mechanism is now well understood for CB1 orthosteric agonists and it shares similarities with other class A GPCRs [CB1 is a GPCR that signals primarily via Gle to Gs , Gq/11 [Gs [q/11 , and G121 [12/13 . Althoug release , G12/13 e growth . Consequ surface . Mutatio A GPCRs ,151,152.On the other hand, the mechanism and function of β-arrestin recruitment by CB1 is less well understood. At the functional level, β-arrestins regulate CB1 desensitization and downregulation in a brain-region-specific manner, which can result in differential tolerance development to cannabinoid effects . An impoThe biased-signaling properties of CB1 orthosteric agonists have been investigated in different in vitro systems. However, common CB1 ligands show little preference for either G-protein or β-arrestin signaling, with conflicting results across different studies ,122,123.In conclusion, CB1 is highly expressed throughout the CNS in excitatory and inhibitory neurons as well as astrocytes, giving it the potential to impact a myriad of CNS physiological functions and disease states. However, this broad expression also limits its utility due to adverse effects and abuse potential. Biased signaling has been suggested as a strategy to dissociate therapeutic effects from the undesired effects of CB1 activity. However, the functional consequences of CB1-biased signaling are still poorly understood due to the lack of signaling specificity of know orthosteric agonists. The development of biased allosteric ligands may be a viable strategy to dissociate the activation of G-proteins, β-arrestin1, or β-arrestin2 and refine CB1 targeted therapeutics.


Despite increasing evidence for an association between circulating uric acid (UA) and depression, the directionality of this association remains unclear and is potentially moderated by low-grade inflammation. Thus, the present study aimed to investigate the cross-sectional association between serum UA concentration and depressive symptoms in Korean individuals with and without low-grade inflammation, as measured using serum high-specific C-reactive protein (hs-CRP) levels. The final study sample comprised 4188 participants, aged 19–79 years, from the Korea National Health and Nutrition Examination Study 2016. Data on serum uric acid (UA) concentrations, serum hs-CRP levels, Patient Health Questionnaire-9 (PHQ-9) scores, and relative covariates were retrieved. Negative binomial regression with adjustment for the complex sample design was used to analyze the associations. After adjusting for covariates, log-transformed serum UA concentrations and total PHQ-9 scores were positively associated  for participants without low-grade inflammation and inversely associated (IRR = 0.64 [95% CI = 0.45–0.92]) for participants with low-grade inflammation. In conclusion, the direction of the association between serum UA and depressive symptoms was the opposite in participants with and without low-grade inflammation. The study has the limitation of potential uncontrolled confounders. However, there is increasing evidence of its association with the central nervous system, including depression2. Most previous studies investigating the association between UA and depression/depressive symptoms reported an inverse association6. However, a recent report on adolescents found that UA levels positively correlated with depression7. UA may have neuroprotective effects, as it is the major antioxidant in human blood8. UA also functions as an intracellular pro-oxidant9, and high serum UA levels are a well-known risk factor for cardiovascular risks11; these are also associated with depression12. The dual anti- and pro-oxidant properties of UA imply that the association between UA and depression may be moderated by tertiary factors.Uric acid (UA), the end product of purine metabolism, is primarily known for its association with gout13. Especially low-grade inflammation has been thoroughly studied as a potential mechanism of mood disorders, including depression15. However, these studies tend to exclude participants with high-level inflammatory states like acute infection, since the symptoms of the acute condition itself can manifest like depression. The role and importance of inflammation in these disorders have been investigated, and recent studies have shown that low-grade inflammation and cardio-metabolic disturbances are associated with mood symptoms17.Inflammation is also known for its association with psychiatric diseases19, albuminuria in type 2 diabetes20, and even mortality21. For instance, low-grade inflammation was shown to significantly amplify the effect of UA on albuminuria in type 2 diabetes, moderating the association20. As UA has potential interactions with low-grade inflammation, its influence on depression might also be moderated by low-grade inflammation, which would further explain previous discrepancies. UA’s anti-and pro-oxidant properties in depression might depend on the level of low-grade inflammation.Recently, numerous studies have focused on the interaction between UA and inflammation in certain conditions, including hypertensionGiven this background, the present study sought to clarify the cross-sectional association between UA and depressive symptoms in the Korean general population. We hypothesized that the association between UA and depressive symptoms would differ between individuals with and without low-grade chronic inflammation, as measured using serum high-specific C-reactive protein (hs-CRP) levels.When compared with those without low-grade inflammation, participants with low-grade inflammation were older, more often current smokers, more often had a history of arthritis, and were less often women, had less household income, less often alcohol users, and less often engaged in aerobic exercise. Additionally, hypertension, diabetes, hypercholesteremia, and obesity were more common in those with low-grade inflammation. When comparing laboratory values, serum aspartate aminotransferase (AST), creatinine, and UA levels were higher in those with low-grade inflammation. Finally, there was no significant difference in total Patient Health Questionnaire-9 (PHQ-9) scores between participants with and without low-grade inflammation.p = 0.10). Among participants without low-grade inflammation, there was a positive association between log-transformed serum UA concentrations and total PHQ-9 scores , after controlling for previous covariates. However, in those with low-grade inflammation, there was an inverse association between log-transformed serum UA concentrations and total PHQ-9 scores .The interaction term between log-transformed serum UA concentrations and the presence of low-grade inflammation was marginally associated with total PHQ-9 scores  quartile of UA levels in their study5.Most previous studies reported an inverse association between UA and depression/depressive symptomsAdditionally, differing results may be due to differing study populations and statistical analysis methods. The present study evaluated depressive symptoms in the general population, while most studies, except the one from China discussed above, enrolled depressed patients. Furthermore, we excluded participants with hs-CRP levels > 10 mg/L. During acute infection, the PHQ-9 may actually reflect acute illness and not depressive symptoms. Finally, unlike previous studies, the present study used a negative binomial regression approach and adjusted for complex sample designs to reflect the distribution of depressive symptom scale scores in the general population. However, direct comparison is limited as previous studies were not stratified by low-grade inflammation.18. Further, this is known to increase indoleamine dioxygenase, the enzyme that catabolizes tryptophan into neurotoxic kynurenine instead of serotonin18, and is also known to cause damage to membrane polysaturated fatty acids and may affect serotoninergic membrane receptors18. Finally, this can also induce neurodegeneration, also a known factor of depression18. In those without low-grade inflammation, the association between UA and depression was reversed. In this situation, UA levels may present a certain pro-oxidant capacity9. The prior study that reported that higher serum UA levels were associated with depression revealed that serotonin levels in the brain were negatively associated with blood UA levels in depressed patients7. Pro-oxidant UA may result from increased nucleic acid oxygenolysis in depression7. However, the pro-oxidant UA effects also induce inflammation22, which CRP may not be able to reflect as the association with oxidative stress differs with inflammatory markers23. Recent meta-analyses reported a significant association between inflammation and mood disorders, including depression15. Inflammation has also been associated with emotional reactivity in patients even in remission16. A recent study revealed that these symptoms are associated with cardiovascular function, glucose metabolism, and lipid metabolism17. These cardiovascular disturbances, components of the metabolic syndrome, are all linked to oxidative stress, inflammation, and depressive symptoms25. Therefore, further investigation of the complex association between oxidative stress, inflammation, and depressive symptoms is needed26.The opposite direction of the associations between UA and depressive symptoms with and without low-grade inflammation may be due to UA’s dual pro- and antioxidant properties. In participants with low-grade inflammation, UA levels may present antioxidant capacity. Lower antioxidant capacity with inflammation induces oxidative and nitrosative stress, which causes damage to various cellular components including fatty acids, proteins, DNA, and mitochondria27. The authors suggested that despite the increased xanthine oxidase activity in depression as a compensatory mechanism to oxidative stress, it is insufficient to overcome the UA consumption as an antioxidant, thus leading to decreased UA levels27. This compensatory mechanism may in fact be dependent on low-grade inflammation. In people with low-grade inflammation, despite the increase in xanthine oxidase, UA use as an antioxidant may decrease its levels. However, in those without low-grade inflammation, a hyperactive purine metabolism in depression could lead to UA accumulation. Further studies investigating the association between specific purine metabolism pathway metabolites and enzymes based on inflammation status in depression may clarify this issue.One additional mechanism that might be considered together with oxidative stress is the purine metabolism pathway. UA is the end product of the purine metabolism; hypoxanthine is catabolized to xanthine, and xanthine to UA by the enzyme xanthine oxidase. A recent study reported an altered purine metabolism in depression and that levels of inosine and guanine, upstream metabolites of xanthine in purine metabolism, were decreased and xanthine levels were increased in patients with depression28. Longitudinal studies are thus needed to clarify causality. Second, our assessment of depressive symptoms was based on the PHQ-9, which utilizes a self-report measure. However, the PHQ-9 is based on the Diagnostic and Statistical Manual of Mental Disorders and has significant internal consistency and test–retest reliability. Third, there may be other covariates that we did not control. Despite controlling for potential confounders, specific symptoms related to depression such as emotional dysregulation16, anxiety3, sleep29, aggression30, and impulsivity31 are also known to be associated with circulating UA or hs-CRP levels. As this study was based on data retrieved from the Korea National Health and Nutrition Examination Study (KNHANES), the confounders we could control were limited. Other potential confounders might have influenced the association. Fourth, an even larger sample would enable further stratifications for specific covariates as an additional analysis, which could expand our knowledge between the association of UA and depressive symptoms. However, a larger sample would increase the risk of type I error. Fifth, the interaction term was only marginally significant, even though the stratification was based on a priori hypothesis. Finally, the KNHANES focuses on depression/depressive symptoms, and the influences of other psychiatric disorders were not evaluated.The present study has some limitations. First, it utilized a cross-sectional design such that causality cannot be determined. Despite controlling for diet and exercise, which can influence UA concentrations, other lifestyle factors associated with depression may influence UA levels. Additionally, participants with higher UA have a higher probability of hypertension, and beta-blockers commonly prescribed for hypertension might have a longitudinal association with depressionDespite its limitations, the present study also has its strengths. First, the data presented here reveal an association between serum UA and depressive symptoms dependent on low-grade inflammatory status, which may explain discrepancies with previous studies. Second, we used a negative binomial regression adjusted for a complex sample design using a national, representative population. These analyses maximized statistical power and reflected the distribution of PHQ-9 scores in the general population.In conclusion, our study revealed an opposite association between serum UA and depressive symptoms in participants with and without low-grade inflammation. This emphasizes the importance of screening for and diagnosing depression in those with serum UA and hs-CRP level imbalances. Further research should use a longitudinal design to clarify the specific mechanisms underlying this relationship and should consider low-grade inflammation as a potential moderator.Data were retrieved from the KNHANES VII-1 (2016), an annual national representative survey of noninstitutionalized civilians in the Republic of Korea. The KNHANES assesses health and nutritional status and estimates the prevalence of chronic diseases. To this end, the KNHANES uses a complex, multi-stage probability sample design. In total, 192 primary sampling units (PSUs) were selected based on administrative districts and housing types in the Republic of Korea. A systemic sampling system with intra-stratification by age, sex, and residential area was applied to extract 23 households from each PSU. Finally, within each household, those aged ≥ 1 year were eligible to participate.32, and those with high-sensitive C-reactive protein (hs-CRP) levels > 10 mg/L indicating an acute infection, yielded 4345 participants. Finally those with missing values for covariates, including those who did not fast ≥ 8 h prior to blood sampling, were excluded. The final sample included 4188 participants.Data from subjects who participated in the health interviews, health examinations, and the nutritional survey from the KNHANES VII-1 (2016) between January and December 2016 were included. Self-administered and structured questionnaires were used to collect information about participants’ demographic, clinical, and nutritional characteristics, and physical examinations were conducted. The total number of participants in the KNHANES VII-1 was 8150, 6703 of whom participated in all three individual surveys. In our study, only participants 19–79 years of age were included, yielding 4943 participants, as the Patient Health Quesionnaire-9 (PHQ-9) was only conducted for those who were ≥ 19-years old. Excluding participants with missing serum UA and PHQ-9 data yielded 4592 participants, and further excluding those who were pregnant/breastfeeding, those who were being treated for depression as medication is also known to affect UA levelsEthical approval for survey procedures by the Institutional Review Board of Korea Centers for Disease Control and Prevention was waived as the KNHANES was conducted to improve national government public welfare measures. All methods were carried out in accordance with relevant guidelines and regulations. Written informed consent was obtained from all survey participants. Ethical approval for the study design by the Institutional Review Board of Seoul National University Hospital was waived due to the anonymity and publicly available nature of the data.33. In four participants, UA levels were below detection limits (< 1.0 mg/dL) and imputed as 0.9 mg/dL. Serum hs-CRP levels were measured by an immunoturbidimetric method using the Cobas analyzer . Levels in 40 participants in the final population were < 0.15 mg/L and imputed as 0.149 mg/L. The target monthly coefficient variances for both UA and hs-CRP were set to ≤ 5% and were satisfied. In our study, the presence of low-grade inflammation was defined as hs-CRP level ≥ 1.0 mg/L, which is usually used as a cutoff to distinguish low from intermediate cardiovascular risk34. This value was also equal to the highest quartile of hs-CRP levels in the final population.In the KNHANES VII-1, after ≥ 8 h of fasting, blood samples were collected to assess levels of biochemical markers. Venous serum samples were collected in a serum separating tube, centrifuged at 3000 rpm for 15 min, and stored at 2–8 °C. Serum UA and hs-CRP were measured within 24 h of sample collection. Serum UA levels were measured by the colorimetric enzymatic (uricase) method using the Hitachi Automatic Analyzer 7600–210 (Hitachi/Japan)36. The primary outcome of the present study was the total PHQ-9 score. It was used as a continuous variable to assess associations with depressive symptom severity and to maintain statistical power.In the KNHANES VII-1, the Korean version of the PHQ-9 was used to assess depressive symptom severity. The PHQ-9 is a nine-item self-report questionnaire. Each question is scored from 0 to 3 points based on the frequency of each symptom. The PHQ-9 has significant internal consistency (α = 0.81–0.86) and test–retest reliability (coefficient = 0.79–0.89)43. We controlled for the following health interview covariates; age (continuous variable), sex (men or women), region , income (less than the 1st quartile of housing income or at least the 1st quartile of housing income), education (not a college graduate or at least a college graduate), unemployment (yes or no), marital status , alcohol use (yes or no), smoking behavior (current smoker or current non-smoker), aerobic exercise (yes or no), history of cancer (yes or no), and history of arthritis . Alcohol use was defined as drinking ≥ 1 cup of alcoholic beverage per month for the past year. A current smoker was defined as someone who is currently smoking and had smoked ≥ 100 cigarettes throughout their lifetime. Aerobic exercise was defined as ≥ 1¼ h per week of high intensity physical activity, or ≥ 2½ h per week of moderate intensity physical activity .Covariates for adjustment were based on previous studies investigating the association between UA and depression/depressive symptoms or known associations with UA or depression/depressive symptoms44, obesity (body mass index ≥ 25 kg/m2)45, and anemia (hemogloblin < 12 g/dL for women and < 13 g/dL for men). The definitions for these conditions were based on the guideline of the KNHANES. All these variables were treated dichotomously. Additionally, serum AST, creatinine, and hs-CRP levels were used as continuous covariates.The following health examination covariates were further controlled for: hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or the use of antihypertensive medication), diabetes , hypercholesteremia 46.Factors from the nutritional survey were also controlled for. These included daily energy and daily protein intake, calculated from a 24-h dietary recall of the day before the nutritional survey (midnight to midnight). Calculations were based on the 8th food composition table by the Korean National Rural Resources Development Institutet tests for continuous variables, with the exception of PHQ-9 scores, which were analyzed using the Kruskal Wallis test. Due to the positive skewness and excess of zeros (37.8%) among participant PHQ-9 scores, we used negative binomial regression to analyze the relationship between serum UA and depressive symptoms. Initially, the statistical significance of the interaction between log-transformed UA and the presence of low-grade inflammation for PHQ-9 data was assessed, controlling for previous covariates. The multivariate analysis was stratified by low-grade inflammation, based on a cutoff value of 1 mg/L of hs-CRP. The multivariate analysis was controlled for age, sex, region, income, education, unemployment, marital status, alcohol use, smoking behavior, aerobic exercise, history of cancer, history of arthritis, hypertension, diabetes, hypercholesteremia, obesity, anemia, serum AST, serum creatinine, serum hs-CRP, daily energy intake, and daily protein intake.Analyses were adjusted for the sampling procedures and weights of the KNHANES complex sample design. For skewed covariates, logarithmic transformations were performed for further analysis. Initially, demographic, clinical, and nutritional characteristics were compared between participants with and without low-grade inflammation. Chi-square tests were performed for categorical variables and We calculated model fitness by dispersion indices. When calculating these dispersions, weights were standardized to the sample size of each stratified group of low-grade inflammation by dividing individuals’ weights by the mean weight of their stratified group.https://www.R-project.org/) was used for the design-based Kruskal Wallis test. Statistical tests were two-sided, and p < 0.05 was considered statistically significant, with the exception for interaction analysis, in which p < 0.10 was considered statistically significant47.Statistical analyses were mainly performed with SSPS Version 23.0 . STATA Version 15.0  was used for the negative binomial regression, and R Version 3.6.2 (


Insular woodiness (IW), referring to the evolutionary transition from herbaceousness toward woodiness on islands, has arisen more than 30 times on the Canary Islands (Atlantic Ocean). One of the IW hypotheses suggests that drought has been a major driver of wood formation, but we do not know in which palaeoclimatic conditions the insular woody lineages originated. Therefore, we provided an updated review on the presence of IW on the Canaries, reviewed the palaeoclimate, and estimated the timing of origin of woodiness of 24 insular woody lineages that represent a large majority of the insular woody species diversity on the Canaries. Our single, broad‐scale dating analysis shows that woodiness in 60%–65% of the insular woody lineages studied originated within the last 3.2 Myr, during which Mediterranean seasonality (yearly summer droughts) became established on the Canaries. Consequently, our results are consistent with palaeoclimatic aridification as a potential driver of woodiness in a considerable proportion of the insular woody Canary Island lineages. However, the observed pattern between insular woodiness and palaeodrought during the last couple of million years could potentially have emerged as a result of the typically young age of the native insular flora, characterized by a high turnover. We provided an updated review on the presence of insular woodiness on the Canaries, reviewed the palaeoclimate, and estimated the timing of origin of woodiness of 24 insular woody lineages of the Canary Islands (Spain). Our single, broad‐scale molecular dating analysis shows that woodiness in 60%–65% of the insular woody lineages studied originated within the last 3.2 Myr, consistent with palaeoclimatic aridification as a potential driver of woodiness. Since reliable fossils for insular woody lineages are lacking on the Canary Islands, this dataset allowed us to build an evolutionary framework in which we can compare the colonization times of the Canary Island lineages using a single analysis based on the same set of fossil calibration points and calibration settings. We modified the Janssens dataset to meet our research question: Eudicots were retained since most insular woody transitions on the Canaries belong to the asterid and rosid clades, and we excluded most of the early diverging angiosperms (no native insular woody lineages) as well as nearly all monocots (no wood development). This reduced our dataset to 23,776 species and an associated 42 fossil calibration points.Janssens et al.  mined a matK and rbcL sequences from 137 Canary Island species that are endemic, native or probable native according to Arechavaleta et al.  DNA Bank of the Canarian Flora and LPA herbarium, housed at the Botanical Garden ‘Viera y Clavijo’ ‐ Unidad Asociada al CSIC, Cabildo de Gran Canaria , (2) herbaria of Naturalis Biodiversity Center , Meise Botanic Garden (Belgium), La Laguna University , and Texas (USA), and (3) botanical gardens of Leiden (The Netherlands), Utrecht (The Netherlands), and Marburg (Germany). Sequences from the remaining species were downloaded from GenBank. Vouchers used in our study are compiled in Table We then included a et al. . Of thesa et al. . We alsoAlthough we were targeting the closest related continental—mainly Mediterranean—species for each Canary Island lineage studied as identified by previous phylogenies Table , it is pConvolvulus, two separate IW shifts were identified on the Canaries and were treated as independent IW events in our analysis (Convolvulus 1: C. scoparius versus Convolvulus 2: C. canariensis‐C. fruticulosus‐C. lopez‐socasii). Two woody Canary lineages of Ononis, one with an insular woody origin (Ononis 1: O. christii) and the other with a continental derived woody origin (Ononis 2: O. hesperia) were also dated independently  has a known continental derived woody origin. A second independent woody Euphorbia lineage (Euphorbia 2: represented by E. mellifera) is included as a derived woody lineage because it is unclear whether the insular woody shift occurred on the Canaries or on adjacent Macaronesian archipelagos .In total, the species investigated belong to 39 independent, single colonization events and 18 flowering plant families: 24 insular woody lineages (representing 94% of the insular woody species diversity on the Canaries as identified in this paper), nine herbaceous eudicot lineages, four derived woody lineages (developed their woodiness outside the Canaries), and two ancestrally woody lineages. For the genus matK‐rbcL phylogeny did not match the previously published phylogenies based on various plastid or nuclear markers. For instance, matK‐rbcL sequences did not discriminate between the Canary Island clade and the continental outgroup . For the Aeonium alliance, we could only provide the dating estimate for the colonization event, because the phylogenetic position of the insular woody Aeonium species were scattered among Aichryson and Monanthes species in our megaphylogeny. For the Canary Island Lobularia, Convolvulus 2 and Ononis 1 and 2 clades, matK and/or rbcL markers of the continental outgroup could not be successfully sequenced. Therefore, we selected the crown node to estimate colonization time  using the KingFisher Flex magnetic particle processor (Thermo Scientific). The primer pair “1R‐KIM”/“3F‐KIM” (forward and reverse) was used for matK amplification . Thermal cycling conditions were as follows: 98°C for 45 s (s), followed by 40 cycles of 98°C for 10 s, 53°C or 66°C for 30 s, 72°C for 40 s, and a final extension at 72°C for 7 min. The PCR products were verified by electrophoresis in 2% agarose e‐gel stained with SYBR™ Safe DNA Gel Stain (Thermo Scientific).For each specimen, a 1 cmmatK and rbcL sequences were added into an existing, aligned angiosperm‐wide dataset  for bidirectional Sanger sequencing. Consensus sequences were generated and edited manually using Geneious version 11.1  to reduce computational time.We used the CIPRES scientific portal for maximum likelihood (ML) phylogeny inference using RAxML—HPC version 8 .We took 42 age constraints across the angiosperm phylogeny from Janssens et al.  see Tab. The rooAeonium was excluded due to phylogenetic inconsistencies in the Aeonium alliance); 19 of these are considered equal since all island species are insular woody . The up3.2A detailed summary of the literature overview of the major palaeoclimatic events during the last 20 Myr for the Canary Islands, North Africa and the Mediterranean region, along with a few global trends, are presented in Table 3.3The colonization of the Canary Islands is inferred to be relatively recent for the clades studied, even with the most conservative dating estimates based on stem ages, with most events occurring in the last 3.2 million years (Myr) for both herbaceous and ancestrally or derived woody lineages  review is the largest dating study of an insular flora, representing 94% of the insular woody species diversity on the Canary Islands. Using a critical re‐evaluation on the occurrence of species that have evolved their woodiness on the Canary Islands, we have identified 196 insular woody species that are the result of at least 35 independent shifts, many of which have originated in the last 3.2 Myr Figure .The reason why a considerable number of species evolved into insular woody shrubs or trees following colonization of the Canaries remains incompletely understood. Based on their current distribution—50% of the extant insular woody Canary Island species are native to markedly dry  regions Table —it is plNotably, our large‐scale two‐gene dating analysis shows that 70% of the dating estimates match previous studies, despite the use of alternative calibration points  and different DNA markers  of the insular woody lineages originated from the early Pliocene onwards Figures . In thisArgyranthemum based on mean stem age  is consistent with MIS 10, which may imply that this relatively short but intense aridification episode could have driven woodiness and subsequent fast radiation in this genus, originating 19 Canary Island species. Without implying a causal relationship, the experimental study by Dória et al. , leading to the spectacular woody, ‘walking stick’ accession . Indeed4.4Aeonium (28 sp.), Sideritis (25 sp.), Limonium (22 sp.), Echium (21 sp.), Micromeria (21 sp.), Argyranthemum (23 sp.), Cheirolophus (18 sp.), Lotus (19 sp.), Sonchus (18 sp.), and Crambe (12 sp.), eight include multiple insular woody species accounting for 143 out of 196 insular woody species on the Canary Islands (73%). In contrast, the herbaceous genera mostly do not harbor more than five species  and their herbaceous continental relatives  resemble each other more, the increased diversification in many insular woody Canary Island lineages may not be in conflict with information in the literature.Although the link between IW and diversification on the Canaries is obvious, identifying the traits that promote diversification is one of the unresolved key questions in plant evolutionary sciences . This is in conflict with the niche pre‐emption hypothesis stating that early colonizers have had more opportunity to occupy the available niches compared to later colonizers, and therefore, older lineages should have had more chance to diversify into larger clades ; Formal analysis (lead); Investigation ; Methodology ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Steven Janssens: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐review & editing (supporting). Vincent Merckx: Formal analysis ; Methodology ; Supervision (supporting); Writing‐review & editing (supporting). Rutger Vos: Formal analysis (supporting); Methodology (supporting); Writing‐review & editing (supporting). Luis Valente: Formal analysis (supporting); Methodology (supporting); Writing‐review & editing (supporting). Alexander Zizka: Data curation ; Formal analysis (supporting); Methodology (supporting); Writing‐review & editing (supporting). Maximilian Larter: Formal analysis (supporting); Methodology (supporting); Writing‐review & editing (supporting). Betül Karabayir: Formal analysis (supporting); Investigation ; Methodology (supporting); Writing‐review & editing (supporting). Daphne Maaskant: Formal analysis (supporting); Investigation ; Methodology (supporting). Youri Witmer: Formal analysis (supporting); Investigation ; Methodology (supporting). José María Fernández‐Palacios: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Writing‐review & editing (supporting). Lea de Nascimento: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Writing‐review & editing (supporting). Ruth Jaén‐Molina: Investigation ; Writing‐review & editing (supporting). Juli Caujapé Castells: Investigation ; Writing‐review & editing (supporting). Águedo Marrero‐Rodríguez: Investigation . Marcelino del Arco: Investigation . Frederic Lens: Conceptualization (lead); Data curation ; Formal analysis ; Funding acquisition (lead); Investigation ; Methodology (lead); Project administration (lead); Supervision (lead); Writing‐original draft (lead); Writing‐review & editing (lead).Fig S1‐S12Click here for additional data file.Fig S13Click here for additional data file.Table S1‐S6Click here for additional data file.Supplementary MaterialClick here for additional data file.


Fluorine is widely dispersed in nature and has multiple physiological functions. Although it is usually regarded as an essential trace element for humans, this view is not held universally. Moreover, chronic fluorosis, mainly characterized by skeletal fluorosis, can be induced by long-term excessive fluoride consumption. High concentrations of fluoride in the environment and drinking water are major causes, and patients with skeletal fluorosis mainly present with symptoms of osteosclerosis, osteochondrosis, osteoporosis, and degenerative changes in joint cartilage. Etiologies for skeletal fluorosis have been established, but the specific pathogenesis is inconclusive. Currently, active osteogenesis and accelerated bone turnover are considered critical processes in the progression of skeletal fluorosis. In recent years, researchers have conducted extensive studies in fields of signaling pathways , stress pathways (oxidative stress and endoplasmic reticulum stress pathways), epigenetics (DNA methylation and non-coding RNAs), and their inter-regulation involved in the pathogenesis of skeletal fluorosis. In this review, we summarised and analyzed relevant findings to provide a basis for comprehensive understandings of the pathogenesis of skeletal fluorosis and hopefully propose more effective prevention and therapeutic strategies. Fluorine is one of the most common halogens, which usually exists in the environment as compounds. Fluorine or fluoride enters the human body primarily through drinking water, food, and air. Once absorbed into the bloodstream, it is easily transported throughout the body in the form of ions. More than 90% of absorbed fluoride is distributed in bone tissue, with the total amount of fluorine in a normal human body being approximately 2.6 g . FluoridFluorine has a strong bone affinity and tends to accumulate in bone tissue leading to skeletal fluorosis. Excessive fluoride will cause a series of damages to osteoblasts, osteoclasts, cartilage tissue, and bone mineralization in humans . FluorinResearchers have made significant progress in understanding the regulation of signaling pathways, oxidative stress, endoplasmic reticulum stress, DNA methylation, and non-coding RNAs in recent years. By analyzing several studies on the relevant signaling pathways, stress pathways, epigenetics, and interaction networks among them involved in the pathogenesis of skeletal fluorosis, this review seeks to deepen understanding of the pathogenesis of fluorosis and hopefully propose more targeted prevention and therapeutic strategies.Wnt is a cytokine involved in various biological processes , and theSun et al. confirmed that fluoride significantly increased osteoblast activity, as evidenced by the observed enhanced osteogenesis in their study . FluoridHere, we review the mechanism of action of the Wnt/β-catenin signaling pathway in skeletal fluorosis, and it is evident that this signaling mediates the enhanced osteogenic effects of fluoride. Moreover, Wnt/β-catenin signaling pathway is connected to many signaling pathways in the control of osteoblast and chondrocyte proliferation and differentiation, such as PIK/Akt and hedgehog (Hh) signaling, and subsequent studies to explore their interactive regulation are even more valuable for comprehension of the pathogenesis of skeletal fluorosis.Notch signaling pathway primarily mediates intercellular interactions and plays an integral part in determining cell fate and function together with the regulation of skeletal homeostasis . The intNotch signaling can inhibit osteoblast differentiation ,31, therNotch signaling pathway displays a significant role in cell proliferation, differentiation, and apoptosis. Nonetheless, there are relatively few investigations on the relationship between Notch signaling pathway and skeletal fluorosis, mainly focusing on the mechanisms that regulate the proliferation and differentiation of osteoblasts. An in-depth investigation of the role of Notch signaling pathway in regulating other bone tissues and cells and its synergistic or antagonistic effects with other signaling pathways is of great significance for the pathogenesis, prevention, and treatment of skeletal fluorosis.mTOR is a serine/threonine-protein kinase that can regulate a variety of biological processes. PI3K/Akt/mTOR signaling pathway is involved in the proliferation and differentiation of osteoblasts, osteoclasts, and chondrocytes ,35,36. ESkeletal fluorosis is characterized by a disrupted dynamic balance between bone resorption and formation and is intimately linked to regulating PI3K/Akt/mTOR signaling pathway, with a critical role in osteoblast proliferation and differentiation as well as chondrocyte autophagy. Understanding more about the changes of PI3K/Akt/mTOR signaling pathway and related signaling molecules in skeletal fluorosis, as well as its interaction with other signaling pathways, will promote a profound understanding of the pathogenesis of endemic fluorosis and provide a basis for the prevention and treatment of skeletal fluorosis.Hh signaling pathway consists of Hh protein, protein receptors Ptched (Ptch) and Smoothened (Smo), and the 5-zinc finger transcription factor Glis  . There aStudies have revealed that fluoride stimulation increases the expression of Ihh in rat osteoblasts, and this signal, mediated by Smo, induces the expression of Runx2 after activating Gli2, which in turn promotes osteoblast proliferation and contributes to osteosclerosis . MoreoveThe above studies suggest that the Hh signaling pathway is closely associated with oxidative stress and apoptosis and regulates chondrocyte ossification and apoptosis in skeletal fluorosis. In addition, the available data reveal that Hh signaling is also related to Wnt/β-catenin, Notch, Bmp, and other signaling pathways, and its specific regulatory mechanisms in the pathogenesis of skeletal fluorosis that damages cartilage tissue remain to be investigated in depth.Over the years, systematic studies of the mechanisms of fluorosis in animals and humans have revealed that many calcium-regulating hormones and cytokines are also involved in mediating abnormal osteogenic and osteolytic bone metabolism in skeletal fluorosis, such as parathyroid hormone (PTH) and transforming growth factor β (TGF-β). PTH is a polypeptide hormone responsible for regulating calcium homeostasis in the body and exerts an active role in regulating bone turnover. Its effects on cells are mediated by the G protein-coupled receptor  expressed by target cells, including osteoblasts, osteoblast precursor cells, and osteocytes, but excluding osteoclasts ,50. TherIn addition, PTH, as one of the important hormones regulating calcium and phosphorus metabolism in the body, exerts a bidirectional regulatory effect on bone metabolism. High doses of PTH promote bone resorption, while at low doses, it stimulates bone formation. Furthermore, animal and clinical studies have revealed that when PTH is given intermittently subcutaneously, it stimulates bone formation, increases bone density in long bones and vertebrae, and improves the bone quality ,54. It fInsulin is a multifunctional protein hormone that acts by binding to the insulin receptor (IR) in most tissues. IR and intracellular signaling pathways are the main components of the insulin signaling pathway . FulzeleIn the pathogenetic progression of skeletal fluorosis, both local signaling pathways and hormones are involved. Activation or inhibition of signaling is not accomplished linearly within the single signaling pathway mentioned above alone, but rather the signaling pathways interact to form an interactive regulatory network that acts to control biological processes in a synergistic or antagonistic manner . Figure PTH can bind to the same receptor (PTH1P) as the parathyroid hormone-related peptide (PTHrP) synthesized by osteoblasts and mediates bone formation and accelerated bone turnover through multiple pathways. And SOST expressed by osteoblasts is a key negative regulator of bone formation ,62 and aBoth Wnt/β-catenin and Ihh signaling pathways control the proliferation and differentiation of osteoblasts and chondrocytes at multiple stages ,44,45,46At present, studies on the relevant signaling pathways involved in skeletal fluorosis are still predominantly single lineage studies. Nevertheless, active osteogenesis and accelerated bone turnover as crucial processes in the progression of skeletal fluorosis are regulated by sophisticated networks of multiple signaling pathways. The profound exploration of the interactive regulatory mechanisms among signaling pathways in skeletal fluorosis will facilitate the development of specific targeted therapeutic measures.The endoplasmic reticulum (ER) is essential for protein synthesis and secretion in eukaryotic cells . When cePERK is a serine/threonine-protein kinase that can be activated by accumulating misfolded or unfolded proteins on the ER . It can To further clarify the role of the PERK signaling pathway, changes in osteogenic and osteolytic gene expression were studied in osteoblastic cell lines before and after PERK gene interference, and the results showed that fluoride stimulated the protein expression of PERK, Nrf2, osteoprotegerin (OPG), and Runx2 in PERK siRNA-transfected cells to a certain extent . The aboOxidative stress is considered to be an essential mechanism in the pathogenesis of fluorosis. Intake of large amounts of fluoride can cause an imbalance between antioxidant defense mechanisms and free radical levels, leaving cells in a state of oxidative stress . In fluoIn addition, fluoride can likewise participate in the regulation of osteoclast proliferation through the oxidative stress pathway. Calcineurin (CaN) is a serine/threonine phosphatase dependent on calcium (Ca) and calmodulin (CaM), which is involved in the regulation of osteoclast differentiation and proliferation . Animal The above studies have illustrated that fluoride can induce osteoblast apoptosis and osteoclast proliferation through the oxidative stress pathway, providing evidence for the pathogenesis of skeletal fluorosis. However, the molecular mechanisms of fluoride effects on osteoclasts are still relatively poorly investigated, and how fluoride affects osteoclast proliferation and differentiation through the oxidative stress pathway has not been comprehensively clarified, and further in-depth studies are necessary. Besides, since both endoplasmic reticulum stress and oxidative stress are associated with the pathogenesis of skeletal fluorosis, further studies are needed to explore the interrelationship between them to provide new insights into the pathogenesis of skeletal fluorosis.DNA methylation is one of the earliest and most common epigenetic modifications, and it plays an important role in the pathogenesis of skeletal fluorosis. P16 protein is a key factor of cell cycle regulation in the G1/S phase, competitively inhibits the cell progression from G1 phase to S phase and blocks abnormal activation of osteoblasts ,102. In An animal experiment has shown that estrogen receptor α (ERα) mRNA expression is enhanced when the promoter methylation level of the ERα gene is inhibited in osteoblasts, thereby promoting osteoblast proliferation and differentiation . In addiFollowing extensive research on the molecular mechanisms of abnormal bone metabolism in skeletal fluorosis at the molecular biology level, the role of microRNAs (miRNAs) is gradually being discovered. miRNAs as non-coding RNAs are post-transcription regulators of gene expression. miRNAs negatively regulate the expression of their target mRNAs and can inhibit the expression of mRNAs . FluoridIt was pointed out that miR-29a promotes the differentiation process of osteoblasts by targeting and inhibiting the expression of Dkk-1 . The speIn summary, skeletal fluorosis is a chronic and progressive endemic disease with severe risks to human health. Exposure to a specific duration or dose of fluoride can alter osteoblasts, osteoclasts, and chondrocytes’ function, differentiation, and proliferation and lead to skeletal fluorosis by disrupting the balance between bone formation and resorption. Signaling pathways, stress pathways, epigenetics, and interactive regulatory mechanisms play crucial roles in the pathogenesis above. With the development of multi-level and multi-faceted studies in recent years, considerable progress has been made at the molecular biological level in understanding the pathogenesis of skeletal fluorosis. Unfortunately, although a great deal of research has been carried out, there are no clear and effective treatments for patients with skeletal fluorosis.The treatment of skeletal fluorosis in traditional Chinese medicine (TCM) focuses on reducing fluoride concentrations in the body and improving autogenous regulatory mechanisms, while western medicine aims to reduce the toxicity of fluoride to the body and repair the damage through specific interventions . StudiesSince there is no specific effective treatment to cure skeletal fluorosis completely, prevention and control of skeletal fluorosis by using safe drinking water and reducing the use of high fluoride coal burning is currently considered the ideal approach . Particu


A rough orientation relationship (OR) was predicted with the minimum values of 2O2S and γ-Fe were calculated on the atomic scale based on a crystallographic study using the first-principles calculation method. The calculations of the interface energy showed that the S-terminated and La(S)-terminated interface structures were more stable. The results of difference charge density, electron localization function (ELF), the Bader charges and the partial density of states (PDOS) study indicated that the La(S)-terminated interface possessed metallic bonds and ionic bonds, and the S-terminated interface exhibited metallic bond and covalent bond characteristics. This work addressed the stability and the characteristics of the La2O2S/γ-Fe interface structure from the standpoint of crystallography and energetics, which provides an effective theoretical support to the study the heterogeneous nucleation mechanism. As a result, La2O2S particles are not an effective heterogeneous nucleation site for the γ-Fe matrix from crystallography and energetics points of view.Rare earth (RE) inclusions with high melting points as heterogeneous nucleation in liquid steel have stimulated many recent studies. Evaluating the potency of RE inclusions as heterogeneous nucleation sites of the primary phase is still a challenge. In this work, the edge-to-edge matching (E2EM) model was employed to calculate the atomic matching mismatch and predict the orientation relationship between La Based on this model, researchers reported the heterogeneous nucleation potency of La/Ce oxides, oxysulfides and sulfides for the primary δ-ferrite and/or primary γ-austenite during steel solidification, examined using the two-dimensional disregistry model [δ-values they obtained were highly scattered. Relevant studies have been reviewed by the authors [δ-value calculation were not defined based on the two-dimensional lattice disregistry model. Yu and co-workers [2O2S and γ-austenite in an Fe-43Ni expansion alloy and reported the lowest δ-value of 5.42% between the 2O2S and the 2O2S for γ-austenite. However, there was no conclusive experimental evidence to verify it. Robert Tuttle’s work [Previous studies showed that adding the rare earth element La/Ce into steels resulted either in a decrease in the proportion of the columnar grains or the average grain size of the equiaxed grains or both in the case of as-cast austenitic ,2,3, ferry model . However authors . The het-workers  calculate’s work ,17,18 sh3 [2O3 [2O3 [2O2S, which is the typical oxysulfide, was selected. The present work aims to clarify the heterogeneous nucleation potency of γ-Fe on La2O2S particles, combining a crystallographic calculation using the edge-to-edge matching model with the first-principles calculations from an energetics point of view.Recently, the edge-to-edge matching (E2EM) model  original3 , La2O3 [3 [2O3 , Al2O3 [2O3 [2O3 , TiC [292O3 [2O3 ) and the2O2S for γ-austenite, the E2EM model was adopted for crystallographic calculations. The interatomic spacing misfit (fr) along the close-packed (CP) or nearly CP matching rows, as well as the interplanar spacing mismatch (fd) of the CP or nearly CP planes between La2O2S and γ-austenite, were obtained based on the crystal structures, lattice parameters and atomic positions in the two phases. 2O2S.To investigate the heterogeneous nucleation potency of La2O2S phase has a primitive hexagonal structure, and each unit cell contains two lanthanum atoms, two oxygen atoms and one sulfur atom. The γ-Fe lattice parameters were [2O2S lattice parameters were [The γ-Fe phase possesses a face-centered cubic (FCC) lattice structure with four atoms in each unit cell. By contrast, the Laers were : a = 0.32O2S/γ-Fe interface. The interaction between ion and valence electrons was described by the projector augmented wave (PAW) [Effective heterogeneous nucleation must satisfy the crystallographic match and interface stability between the nucleating agent and the nucleating phase. The plane-wave, pseudo-potential density functional theory (DFT) method implemented in the VASP package  was usedve (PAW) . The excve (PAW)  and the ve (PAW) . Due to ve (PAW) . Since Fve (PAW) . 2O2S and γ-Fe were investigated, and the results were compared with published experimental data. To estimate the necessary layer numbers of bulk-like slabs N used in the subsequent interface systems, the surface properties of slabs with different layer numbers were studied. The interface properties of different atomic terminals, including adhesion work, interface energy and interface bonding properties, were calculated to further investigate the interface stability.In order to verify the methodology proposed in this work, the bulk properties of Lacut was 450 eV. In the γ-Fe and La2O2S bulk phase optimization, the Brillouin zone integral was divided by 9 × 9 × 9 and 10 × 10 × 5 Monkhorst-Pack k-point grids (centered on the gamma point), respectively, which was proven to be sufficient in our convergence test. The k-points sampling grids of the surface and interface were generated in the Monkhorst-Pack method of 0.04 grid density by VASPKIT [−4 eV/atom, and the residual Hellmann–Feynman force on each relaxed atom reached 0.03 eV/Å [In the structural relaxation, the plane-wave cutoff E VASPKIT . A vacuu VASPKIT ,28,39, a.03 eV/Å ,28,29.2O2S were obtained, as shown in r and fd between La2O2S and γ-Fe were calculated, as listed in r was 9.43%, and the minimum fd was 4.58%.Based on crystal structures, lattice parameters and atom positions, the E2EM calculations of the CP or nearly CP rows and the CP or nearly CP planes of γ-Fe and La2O2S and γ-Fe could be predicted based on the matching directions and matching planes. The predicted rough OR between La2O2S and γ-Fe could be expressed as r and fd were 9.43% and 20.72%, respectively. Based on this orientation relationship, the properties of the heterogeneous interface between La2O2S and γ-Fe were calculated by the first principles. The effectiveness of La2O2S as the heterogeneous nucleation site of γ-Fe was discussed from the point of view of energy.Because the matching rows must be on the corresponding matching planes, a favorable orientation relationship (OR) between La2O2S lattice constants (a and c), bulk module (B0) and phase equilibrium volume per atom (V0) by LDA and GGA-PBE function. For γ-Fe bulk, the calculated constants by GGA-PBE function were a = b = c = 3.636 Å, 0.5% below the experimental values [2O2S phase, the calculated lattice constants were a = b = 4.134 Å, and c = 7.073 Å. They are roughly consistent with the experimental values [l values  a = b = l values  a = b = 2O2S2 when the surface model contained more than five atomic layers. This figure is close to the experimental value 1.95 J/m2 reported in ref. [In view of the lattice match of the interface, γ-Fein refs. ,28,37. Tin refs. :(1)Esurf in ref.  Therefor2O2SFor the La2O2S2O2S surface have significant impact on the interfacial stability. Based on the surface convergence test result, the stability of different terminal surfaces was investigated. For surface energy of La2O2S, characterized by non-stoichiometry, La, O and S chemical potentials needed to be considered comprehensively to accurately determine the stability in the experimental environment. The surface energy of La2O2S was calculated by Equation (2) [2O2S slab, The atomic species of Lation (2) ,27,28,422O2S slab in equilibrium with those of bulk La2O2S, the relationship between the chemical potential of La2O2S bulk 2 gas and S bulk μLa2O2O2S bulk per formula unit (f.u.); and 2O2S bulk as a heterogeneous nucleus, the actual solidification process was in a S-rich environment, so the S chemical potential was approximated to zero (Based on Equations (3), (5) and (7), the surface energy (Equation (2)) can be rewritten as:The formation heat 2O2S2~−6.29 J/m2. By contrast, the surface energy of La(O)-terminated, O(La)-terminated and S-terminated surfaces remained constant at 4.79 J/m2, 0.68 J/m2 and 0.06 J/m2, respectively. It should be pointed out that the constant value was due to the zero value of (2O2S and γ-Fe were constructed. They were denoted by La(O)-terminated, La(S)-terminated, O(La)-terminated, O(O)-terminated and S-terminated La2O2S/γ-Fe interfaces, as shown in 2O2S2O2S/γ-Fe interface. For interfacing the close-packed planes, the slabs were rotated about an axis normal to the interface so as to align the close-packed directions, resulting in an orientation relationship of 2O2S and γ-Fe was used. The interface structures were fully relaxed in all dimensions by minimizing the Hellmann-Feynman force on every atom.Based on the surface convergence test results and the orientation relationship predicted by edge-to-edge matching crystallography theory, five interface models between Laximation  was invo2O2S and γ-Fe slabs in the same interface system when one of the slabs is kept and other is replaced with vacuum, respectively; and Binding strength of the interface can be predicted by the work of adhesion (ion (11) ,27,28,302O2S/γ-Fe interfaces. The 2O2S/γ-Fe interfaces in descending order was: S-terminated > La(S)-terminated > La(O)-terminated > O(O)-terminated > O(La)-terminated. This indicates that the S-terminated interface had the largest binging strength, followed by the La(S)-terminated interface. This partly resulted from the bonding type between the interface atoms. In addition, the S-terminated and La(S)-terminated surface structures were more stable than others with a smaller surface energy. These demonstrate that it is more likely that the S-terminated and La(S)-terminated interfaces are maintained.2O2S/γ-Fe was calculated from the viewpoint of thermodynamics, and the interface stability of the different terminated interface configurations was quantitatively analyzed. The results can provide theoretical data for the heterogeneous nucleation mechanism of La2O2S on the γ-Fe substrate. According to the calculation method provided in refs. [2O2S and γ-Fe free surfaces in the same interface system; and the other parameters are consistent with those in Equation (8).In addition to the work of adhesion -terminated interfaces, which have smaller interface energy, were more stable than others. It, therefore, can be concluded that S-terminated and La(S)-terminated interfaces are most likely to form. Within the range of The bonding strength of the interface is closely related to the bonding type of the interface atoms, as well as the electronic structure of the interface. To better understand interface atomic bonding, the difference charge density, the electron localization function (ELF), the Bader charges and the partial density of states (PDOS) were employed. The electronic structure and bonding of the more stable S-terminated and La(S)-terminated interface structures were compared to explore the interface stability.2O2S/γ-Fe interface system; and 2O2S slab and γ-Fe slab in the same interface structure, respectively.The difference charge density can be used to analyze the transfer of interatomic charge. The charge accumulation and depletion regions were ascertained. The charge density difference was calculated by the equation below ,30:(13)ΔThe localized distribution characteristics of electrons can be characterized by electron localization function (ELF), which can also confirm the bond types. The ELF was calculated by Equation (14) :(14)ELF=3. Chemical bonds were formed between the different types of interface atoms, and the charge was redistributed after the interface was formed. As shown in To further investigate the bonding characteristics, the partial density of states (PDOS) was also calculated, as shown in r and fd values were both less than 10 pct in an as-cast light alloy based on the E2EM model [2O2S and γ-Fe, the minimum values of fr and fd in construction of the OR were 9.43% and 20.72%, respectively. This implies that, from a crystallographic point view, La2O2S is not an effective nucleant for the nucleation of γ-Fe.To ensure sufficient atomic matching to retain a coherent or semi-coherent interface between the two phases, empirical criteria of the fEM model ,19,20,222O2S the effective heterogeneous nucleus of γ-Fe during the solidification, the solid–solid interface energy of La2O2S/γ-Fe should be between 0 J/m2 and 0.204 J/m2 from the viewpoint of thermodynamics [2 [2O2S/γ-Fe could not meet the interface energy requirements as a heterogeneous nucleation interface. It did not comply with the thermodynamic conditions that make La2O2S particles effective as potent heterogeneous nucleation substrates for the γ-Fe matrix. In addition, factors, such as the size and distribution of La2O2S particles, also impact whether La2O2S can be an effective heterogeneous nucleus of γ-Fe during solidification. Future research will focus on the impact of such factors. The orientation relationship of crystallography will be verified by both electron backscatter diffraction and transmission electron microscopy.Although all of the first-principles calculations were performed at 0 K, the results proved the experimental phenomena of solid-phase systems and solid–liquid interfaces at high temperatures ,47. To mdynamics , for whiamics [2 , as show2O2S particles and the γ-Fe matrix, the values of the interatomic spacing misfit and the interplanar spacing mismatch of which were 9.43% and 20.72%, respectively. This OR was With the crystallographic calculations using the E2EM model, a rough HCP/FCC orientation relationship (OR) was predicted between La2O2S2, followed by the La(S)-terminated interface, which was 3.372 J/m2.For γ-Fe/LaAccording to the bonding analysis of the electronic structure and charge transfer on the two different interfaces, the interface interaction of the S-terminated interface exhibited metallic and ionic bonds, whereas the La(S)-terminated interface exhibited metallic and covalent bonds.2O2S as a nucleation site for γ-Fe. The first-principles calculations revealed that the interfacial energy of La2O2S/γ-Fe cannot meet the interface energy requirements as a heterogeneous nucleation interface. Thus, La2O2S particles are noneffective heterogeneous nucleation substrates for a γ-Fe matrix from crystallography and thermodynamics viewpoints.Crystallographic calculations indicated that the interatomic spacing misfit and the interplanar spacing mismatch were large enough, compared with other reported efficient nucleating systems, leading to weak potency of La