Isolation and structural characterization of exopolysaccharide from the Cordyceps cicadae and the immunomodulatory activity on RAW264.7 cells.

A new exopolysaccharide component named as PC-EPS was isolated from Cordyceps cicadae, and its structure was determined. PC-EPS was identified to be constituted of mannose, glucose, and galactose (28.84:1:19.42), with an average molecular weight of 3.72 × 106  Da, according to the results of monosaccharide composition, Fourier transform infrared, nuclear magnetic resonance, periodate oxidation and Smith degradation, and methylation studies. According to structural characterization, PC-EPS's connection type was made up of →6) -α-d-Manp (1→, →2) -ß-d-Manp (1→, →4) -α-d-Manp (1→, →2) -α-d-Galf (1→, and →4) -α-d-Galp (1→. PC-EPS may significantly increase phagocytosis and RAW264.7 cell proliferation. Additionally, by boosting intracellular lysozyme, cellular acid phosphatase, and cellular superoxide dismutase enzyme concentrations, as well as by promoting the generation of cellular NO, it is the potential to regulate the immunological activity of RAW264.7 cells. Additionally, the effects of PC-EPS on RAW264.7 cells increased their capacities to create tumor necrosis factor-α and interleukin 6 cytokines, all of which suggested that PC-EPS had the potential to improve immunomodulatory activity.

[Clinical efficacy of Xuanju compound capsule combined with urofollitropin in the treatment of patients with idiopathic oligoasthenozoospermia].

OBJECTIVE: To evaluate the therapeutic effects of Xuanju compound capsule combined with urofollitropin (uFSH) in the treatment of idiopathic oligoasthenozoospermia. METHODS: From June 2022 to June 2023, patients with idiopathic oligoastheospermia were enrolled in this study, and divided into trail group (Xuanju compound capsule combined with urofollitropin tablets, n=53) and control group (urofollitropin tablets, n=61) according to the difference in treatment. Treatment methods: Xuanju compound capsule, 3 pills, three times a day; Urofollitropin, 75IU, one times three day. The curses of treatments for control group and trail group is 12 weeks. In order to evaluate the therapeutic effects of control group and trial group, semen volume, sperm concentration, progressive sperm ratio (PR), peripheral serum sex hormone, liver functions were analyzed before and after treatment for two times. RESULTS: Compared with the baseline, the semen volume and liver function were not significantly changed after the treatment in control group and trial group. However, sperm concentration, PR, testosterone (T) levels, follicle stimulating hormone (FSH) levels, and luteinizing hormone (LH) levels were significantly unregulated after the treatment in control group and trial group. More importantly, compared to control group, sperm concentration, PR, T leves, FSH levels, LH levels, and T/E2 ratio of trial group were further enhanced after the treatment, which were statistically significant (P < 0.05). CONCLUSIONS: Xuanju compound capsule combined with urofollitropin tablets could significantly improve the semen quality, up-regulate the testosterone levels and T/E2 ratio in patients with idiopathic oligoasthenozoospermia.

[Liver targeting of compound liposomes mediated by glycyrrhetinic acid derivative receptor and its effect on hepatic stellate cells].

The 3-succinate-30-stearyl glycyrrhetinic acid(18-GA-Suc) was inserted into glycyrrhetinic acid(GA)-tanshinone Ⅱ_A(TSN)-salvianolic acid B(Sal B) liposome(GTS-lip) to prepare liver targeting compound liposome(Suc-GTS-lip) mediated by GA receptors. Next, pharmacokinetics and tissue distribution of Suc-GTS-lip and GTS-lip were compared by UPLC, and in vivo imaging tracking of Suc-GTS-lip was conducted. The authors investigated the effect of Suc-GTS-lip on the proliferation inhibition of hepatic stellate cells(HSC) and explored their molecular mechanism of improving liver fibrosis. Pharmacokinetic results showed that the AUC_(Sal B) decreased from(636.06±27.73) µg·h·mL~(-1) to(550.39±12.34) µg·h·mL~(-1), and the AUC_(TSN) decreased from(1.08±0.72) µg·h·mL~(-1) to(0.65±0.04) µg·h·mL~(-1), but the AUC_(GA) increased from(43.64±3.10) µg·h·mL~(-1) to(96.21±3.75) µg·h·mL~(-1). The results of tissue distribution showed that the AUC_(Sal B) and C_(max) of Sal B in the liver of the Suc-GTS-lip group were 10.21 and 4.44 times those of the GTS-lip group, respectively. The liver targeting efficiency of Sal B, TSN, and GA in the Suc-GTS-lip group was 40.66%, 3.06%, and 22.08%, respectively. In vivo imaging studies showed that the modified liposomes tended to accumulate in the liver. MTT results showed that Suc-GTS-lip could significantly inhibit the proliferation of HSC, and RT-PCR results showed that the expression of MMP-1 was significantly increased in all groups, but that of TIMP-1 and TIMP-2 was significantly decreased. The mRNA expressions of collagen-I and collagen-Ⅲ were significantly decreased in all groups. The experimental results showed that Suc-GTS-lip had liver targeting, and it could inhibit the proliferation of HSC and induce their apoptosis, which provided the experimental basis for the targeted treatment of liver fibrosis by Suc-GTS-lip.

A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer.

According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell research.

A nitric oxide burst at the shoot apex triggers a heat-responsive pathway in Arabidopsis.

When confronted with heat stress, plants depend on the timely activation of cellular defences to survive by perceiving the rising temperature. However, how plants sense heat at the whole-plant level has remained unanswered. Here we demonstrate that shoot apical nitric oxide (NO) bursting under heat stress as a signal triggers cellular heat responses at the whole-plant level on the basis of our studies mainly using live-imaging of transgenic plants harbouring pHsfA2::LUC, micrografting, NO accumulation mutants and liquid chromatography-tandem mass spectrometry analysis in Arabidopsis. Furthermore, we validate that S-nitrosylation of the trihelix transcription factor GT-1 by S-nitrosoglutathione promotes its binding to NO-responsive elements in the HsfA2 promoter and that loss of function of GT-1 disrupts the activation of HsfA2 and heat tolerance, revealing that GT-1 is the long-sought mediator linking signal perception to the activation of cellular heat responses. These findings uncover a heat-responsive mechanism that determines the timing and execution of cellular heat responses at the whole-plant level.

Lipidomic Remodeling in Begonia grandis Under Heat Stress.

Characterization of the alterations in leaf lipidome in Begonia (Begonia grandis Dry subsp. sinensis) under heat stress will aid in understanding the mechanisms of stress adaptation to high-temperature stress often occurring during hot seasons at southern areas in China. The comparative lipidomic analysis was performed using leaves taken from Begonia plants exposed to ambient temperature or heat stress. The amounts of total lipids and major lipid classes, including monoacylglycerol (MG), diacylglycerol (DG), triacylglycerols (TG), and ethanolamine-, choline-, serine-, inositol glycerophospholipids (PE, PC, PS, PI) and the variations in the content of lipid molecular species, were analyzed and identified by tandem high-resolution mass spectrometry. Upon exposure to heat stress, a substantial increase in three different types of TG, including 18:0/16:0/16:0, 16:0/16:0/18:1, and 18:3/18:3/18:3, was detected, which marked the first stage of adaptation processes. Notably, the reduced accumulation of some phospholipids, including PI, PC, and phosphatidylglycerol (PG) was accompanied by an increased accumulation of PS, PE, and phosphatidic acid (PA) under heat stress. In contrast to the significant increase in the abundance of TG, all of the detected lysophospholipids and sphingolipids were dramatically reduced in the Begonia leaves exposed to heat stress, suggesting that a very dynamic and specified lipid remodeling process is highly coordinated and synchronized in adaptation to heat stress in Begonia plants.

Anlotinib plus Epirubicin Followed by Anlotinib Maintenance as First-line Treatment for Advanced Soft-tissue Sarcoma: An Open-label, Single-arm, Phase II Trial.

PURPOSE: The treatment outcome for locally advanced or metastatic soft-tissue sarcoma (STS) remains unsatisfactory. Anlotinib had demonstrated impressive activity in the subsequent-line treatment of STS. This study investigated the combination of anlotinib and epirubicin followed by anlotinib maintenance as first-line treatment for patients with advanced STS. PATIENTS AND METHODS: This prospective, open-label, single-arm, phase II trial was conducted in Zhongshan Hospital, Fudan University. Eligible patients were ages 18 years or older and had previously untreated, pathologically confirmed, unresectable locally advanced or metastatic STS. All patients received up to six cycles of anlotinib plus epirubicin followed by anlotinib maintenance until disease progression, unacceptable toxicity, or death. The primary endpoint was the progression-free survival (PFS) rate at 6 months. The study was registered on chictr.org (identifier ChiCTR1900024928). RESULTS: From June 2019 to August 2020, 30 patients were enrolled. By December 2021, the median PFS was 11.5 months [95% confidence interval (CI): 8.6-14.4 months], while the median overall survival was not reached (95% CI: NE-NE). The objective response rate was 13.33% and the disease control rate was 80.0%. The most common adverse events (AE) included anemia (43.3%), nausea/vomiting (40.0%), fatigue (36.7%), leukopenia (30.0%), and proteinuria (10.0%), which were mainly of grade 1 or 2. The most frequent grade 3 or 4 AEs were anemia (10.0%), febrile neutropenia (33.3%), hypothyroidism (3.3%), and leukopenia (3.3%). No treatment-related death occurred. CONCLUSIONS: The combination of anlotinib and epirubicin followed by anlotinib maintenance demonstrated promising efficacy with a favorable safety profile.

Regulation of Calvin-Benson cycle enzymes under high temperature stress.

The Calvin-Benson cycle (CBC) consists of three critical processes, including fixation of CO2 by Rubisco, reduction of 3-phosphoglycerate (3PGA) to triose phosphate (triose-P) with NADPH and ATP generated by the light reactions, and regeneration of ribulose 1,5-bisphosphate (RuBP) from triose-P. The activities of photosynthesis-related proteins, mainly from the CBC, were found more significantly affected and regulated in plants challenged with high temperature stress, including Rubisco, Rubisco activase (RCA) and the enzymes involved in RuBP regeneration, such as sedoheptulose-1,7-bisphosphatase (SBPase). Over the past years, the regulatory mechanism of CBC, especially for redox-regulation, has attracted major interest, because balancing flux at the various enzymatic reactions and maintaining metabolite levels in a range are of critical importance for the optimal operation of CBC under high temperature stress, providing insights into the genetic manipulation of photosynthesis. Here, we summarize recent progress regarding the identification of various layers of regulation point to the key enzymes of CBC for acclimation to environmental temperature changes along with open questions are also discussed.

Mineralization of cyanides via a novel Electro-Fenton system generating •OH and •O2.

Traditional methods of cyanides' (CN-) mineralization cannot overcome the contradiction between the high alkalinity required for the inhibition of hydrogen cyanide evolution and the low alkalinity required for the efficient hydrolysis of cyanate (CNO-) intermediates. Thus, in this study, a novel Electro-Fenton system was constructed, in which the free cyanides released from ferricyanide photolysis can be efficiently mineralized by the synergy of •OH and •O2-. The complex bonds in ferricyanide (100 mL, 0.25 mM) were completely broken within 80 min under ultraviolet radiation, releasing free cyanides. Subsequently, in combination with the heterogeneous Electro-Fenton process, •OH and •O2- were simultaneously generated and 92.9% of free cyanides were transformed into NO3- within 120 min. No low-toxic CNO- intermediates were accumulated during the Electro-Fenton process. A new conversion mechanism was proposed that CN- was activated into electron-deficient cyanide radical (•CN) by •OH, and then the •CN intermediates reacted with •O2- via nucleophilic addition to quickly form NO3-, preventing the formation of CNO- and promoting the mineralization of cyanide. Furthermore, this new strategy was used to treat the actual cyanide residue eluent, achieving rapid recovery of irons and efficient mineralization of cyanides. In conclusion, this study proposes a new approach for the mineralization treatment of cyanide-containing wastewater.

Role of the transcription factor NFIC in odontoblast gene expression.

The transcription factor NFI-C is essential for root development. Mice lacking NFI-C develop abnormal roots and lose their teeth, resembling radicular dentin dysplasia I in humans. The purpose of this study was to understand the role of NFI-C in dentinogenesis. The authors found statistically significant increases in the expression of several mRNAs in cells lacking NFI-C, suggesting that these molecules might interfere with odontoblast cell migration and differentiation, and consequently with root development.

SPL6 represses signalling outputs of ER stress in control of panicle cell death in rice.

Inositol-requiring enzyme 1 (IRE1) is the most conserved transducer of the unfolded protein response that produces either adaptive or death signals depending on the amplitude and duration of its activation. Here, we report that SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 6 (SPL6)-deficient plants displayed hyperactivation of the endoplasmic reticulum (ER) stress sensor IRE1, leading to cell death in rice panicles, indicating that SPL6 is an essential survival factor for the suppression of persistent or intense ER stress conditions. Importantly, knockdown of the hyperactivated mRNA level of IRE1 rescues panicle apical abortion in the spl6-1 transgenic plants harbouring the IRE1-RNAi constructs, establishing the genetic linkage between the hyperactivation of IRE1 and cell death in spl6-1. Our findings reveal a novel cell survival machinery in which SPL6 represses the transcriptional activation of the ER stress sensor IRE1 in control of ER stress signalling outputs that hinge on a balance between adaptive and death signals for determining cell fates during ER stress.

Circulating MicroRNA-4739 May Be a Potential Biomarker of Critical Limb Ischemia in Patients with Diabetes.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease, which is common but rarely diagnosed. Noninvasive biomarkers are urgently required to assist in the diagnosis of CLI. Accumulating evidence indicates that miRNAs play an important role in the development of various diseases. In this study, microarray profiling revealed 11 miRNAs with significantly altered expression in four T2DM patients with CLI compared with that in four sex- and age-matched T2DM patients without CLI. In independent cohorts, qRT-PCR validation confirmed the increased miRNA-4739 level in patients with CLI versus patients without CLI. miRNA-4739 levels increased with FPG and HbA1c (all P < 0.05). After adjusting for the risk factors, miRNA-4739 levels were found to be associated with an increased odds ratio (OR) of T2DM with CLI (OR =12.818, 95% confidence intervals (CI) 1.148 to 143.143, P = 0.038). ROC curve analysis revealed that the area under the curve (AUC) of miR-4739+confounding risk factors was 0.94 (95% CI 0.891 to 0.998, P < 0.001), which was higher than that of confounding risk factors (AUC 0.94 vs. 0.91, 95% CI -0.122 to 0.060, P > 0.05) and of miR-4739 (AUC 0.94 vs. 0.69, 95% CI -0.399 to -0.101, P < 0.001), respectively. We conclude that elevated plasma miRNA-4739 levels are independently associated with CLI in T2DM patients. miRNA-4739 is implicated as a novel diagnostic marker and a potential therapeutic target for CLI in diabetes.

Expression of aromatase and estrogen receptors in lumbar motoneurons of mice.

Estrogen exerts protective roles in amyotrophic lateral sclerosis (ALS). However, the expression of aromatase (ARO) and estrogen receptors (ERs) in the motoneurons of spinal cord, has not yet been elucidated. By immunohistochemistry, we found that ARO and ERs were present in the ventral horn of adult mice lumbar spinal cord, and colocalized with SMI-32, a motoneuron specific marker. Within motoneurons, we observed that ARO is detected primarily in the cytoplasm, with fewer ARO in the nucleus; ERα and ERß mainly localized in the nucleus with less in the cytoplasm; while GPR30 is located in soma and processes. In conclusion, we found that ERs and ARO are expressed in the motoneurons of lumbar spinal cord in adult mice. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in ALS.

A very rare case of trisomy 4q32.3-4q35.2 and trisomy 21q11.2-21q22.11 in a patient with recombinant chromosomes 4 and 21.

We report the case of a patient with a clinical phenotype consistent with Down Syndrome (DS) who has a novel karyotypic abnormality. Karyotypic analyses were performed to investigate the cause of two spontaneous abortions. A balanced translocation between chromosomes 4 and 21 was identified, along with an additional abnormal chromosome 21. We performed high-resolution banding, comparative genomic hybridization (CGH), and FISH studies in both the patient and her mother to define the abnormality and determine its origin. CGH revealed a gain in copy number on the long arm of chromosome 4, spanning at least 24.4 Mb, and a gain in copy number on the long arm of chromosome 21, spanning at least 16.2 Mb. FISH analysis using a chromosome 21 centromere probe and chromosome 4 long arm telomere (4pter) probe confirmed the origin of the marker chromosome. It has been confirmed by the State Key Laboratory of Medical Genetics of China that this is the first reported instance of the karyotype 47,XX,t(4;21)(q31.3;q11.2),+der(21)t(4;21)mat reported in the world.

Variations in the MHC region confer risk to esophageal squamous cell carcinoma on the subjects from high-incidence area in northern China.

BACKGROUND: The human major histocompatibility complex (MHC) is the most important region in vertebrate genome, and is crucial in innate immunity. Recent studies have demonstrated the possible role of polymorphisms in the MHC region to high risk for esophageal squamous cell carcinoma (ESCC). Our previous genome-wide association study (GWAS) has indicated that the MHC region may confer important risk loci for ESCC, but without further fine mapping. The aim of this study is to further identify the risk loci in the MHC region for ESCC in Chinese population. METHODS: Conditional logistic regression analysis (CLRA) was performed on 24 single nucleotide polymorphisms (SNPs) within the MHC region, which were obtained from the genetically matched 937 cases and 692 controls of Chinese Han population. The identified promising SNPs were further correlated with clinical and clinicopathology characteristics. Immunohistochemistry was performed to explore the protein expression pattern of the related genes in ESCC and neighboring normal tissues. RESULTS: Of the 24 promising SNPs analyzed, we identified three independent SNPs in the MHC region associated with ESCC: rs35399661 (P = 6.07E-06, OR = 1.71, 95%CI = 1.36-2.17), rs3763338 (P = 1.62E-05, OR = 0.63, 95%CI = 0.50-0.78) and rs2844695 (P = 7.60E-05, OR = 0.74, 95%CI = 0.64-0.86). These three SNPs were located at the genes of HLA-DQA1, TRIM27, and DPCR1, respectively. Further analyses showed that rs2844695 was preferentially associated with younger ESCC cases (P = 0.009). The positive immunostaining rates both for HLA-DQA1 and TRIM27 were much higher in ESCC tissues than in neighboring normal tissues (69.4% vs. 26.8% for HLA-DQA1 and 77.6% vs. 47.8% for TRIM27, P<0.001). Furthermore, the overexpression of HLA-DQA1 is correlated significantly with age (P = 0.001) and family history (P<0.001). CONCLUSION: This study for the first time provides evidence that multiple genetic factors within the MHC region confer risk to ESCC on the subjects from high-risk area in northern China.

Synbindin in extracellular signal-regulated protein kinase spatial regulation and gastric cancer aggressiveness.

BACKGROUND: The molecular mechanisms that control the aggressiveness of gastric cancer (GC) remain poorly defined. Here we show that synbindin contributes to the aggressiveness of GC by activating extracellular signal-regulated protein kinase (ERK) signaling on the Golgi apparatus. METHODS: Expression of synbindin was examined in normal gastric mucosa (n = 44), intestinal metaplastic gastric mucosa (n = 66), and GC tissues (n=52), and the biological effects of synbindin on tumor growth and ERK signaling were detected in cultured cells, nude mice, and human tissue samples. The interaction between synbindin and mitogen-activated protein kinase kinase (MEK1)/ERK was determined by immunofluorescence and fluorescence resonance energy transfer assays. The transactivation of synbindin by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was detected using luciferase reporter assay and chromatin immunoprecipitation. RESULTS: High expression of synbindin was associated with larger tumor size (120.8 vs 44.8 cm(3); P = .01), advanced tumor node metastasis (TNM) stage (P = .003), and shorter patient survival (hazard ratio = 1.51; 95% confidence interval [CI] = 1.01 to 2.27; P = .046). Synbindin promotes cell proliferation and invasion by activating ERK2 on the Golgi apparatus, and synbindin is directly transactivated by NF-κB. Synbindin expression level was statistically significantly higher in human GCs with activated ERK2 than those with low ERK2 activity (intensity score of 11.5, 95% CI = 10.4 to 12.4 vs intensity score of 4.6, 95% CI 3.9 to 5.3; P < .001). Targeting synbindin in xenograft tumors decreased ERK2 phosphorylation and statistically significantly reduced tumor volume (451.2mm(3), 95% CI = 328.3 to 574.1 vs 726.1mm(3), 95% CI = 544.2 to 908.2; P = .01). CONCLUSIONS: Synbindin contributes to malignant phenotypes of GC by activating ERK on the Golgi, and synbindin is a potential biomarker and therapeutic target for GC.

Amelogenin and ameloblastin show growth-factor like activity in periodontal ligament cells.

Enamel proteins, particularly amelogenin, have been associated with other functions in addition to regulating enamel biomineralization. Extracts of enamel proteins are currently being used to regenerate periodontal tissues, and new studies suggest that enamel proteins might have chondrogenic and osteogenic properties. In this study, we wanted to determine the effect, if any, of purified recombinant amelogenin and ameloblastin on the adhesion, proliferation, and differentiation of periodontal ligament cells in vitro. Immortomouse-derived periodontal ligament (PDL) cells were grown under permissive and differentiation conditions in the presence of different concentrations of mouse recombinant amelogenin, recombinant ameloblastin, or both. Cells were collected after 4 h to determine attachment, after 24 h to determine proliferation, and after 7, 14, 21 and 28 d to determine differentiation using reverse transcription-polymerase chain reaction (RT-PCR). Both amelogenin and ameloblastin had a small, but statistically significant, effect on increasing the cell attachment and proliferation of PDL cells. Both amelogenin and ameloblastin modulated bone morphogenetic protein (BMP) expression, down-regulated the expression of collagen type I, and induced the de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein. These results suggest that amelogenin, as well as ameloblastin, might have some 'growth factor' activity during periodontium development and regeneration.

Interaction between the enamel matrix proteins amelogenin and ameloblastin.

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.

Role of Hertwig's epithelial root sheath cells in tooth root development.

During tooth development, after the completion of crown formation, the apical mesenchyme forms the developing periodontium while the inner and outer enamel epithelia fuse below the level of the crown cervical margin to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS). The role of HERS cells in root formation is widely accepted; however, the precise function of these cells remains controversial. Functions suggested have ranged from structural (subdivide the dental ectomesenchymal tissues into dental papilla and dental follicle), regulators of timing of root development, inducers of mesenchymal cell differentiation into odontoblasts and cementoblasts, to cementoblast cell precursors. The characterization of the HERS phenotype has been hindered by the small amount of tissue present at a given time during root formation. In this study, we report the establishment of an immortal HERS-derived cell line that can be maintained in culture and then induced to differentiate in vitro. Characterization of the HERS phenotype using reverse transcriptase-polymerase chain reaction and Western blot immunostaining suggests that HERS cells initially synthesize and secrete some enamel-related proteins such as ameloblastin, and then these cells appear to change their morphology and produce a mineralized extracellular matrix resembling acellular cementum. These studies suggest that the acellular and cellular cementum are synthesized by two different types of cells, the first one by HERS-derived cementoblasts and the later by neural crest-derived cementoblasts.