Search

This study aimed to investigate the effect of root dilaceration on the closed-eruption technique treatment and prognosis on impacted immature maxillary central incisors.In this retrospective study, we compared the age at the beginning of the treatment, the treatment duration, root development, and alveolar bone mass after the closed-eruption technique between the impacted immature maxillary central incisors with dilacerated roots (group 1) and those with straight roots (group 2).The mean age at the time of the surgery of group 1 was 0.9 years younger than that of group 2 (P = 0.008). The mean traction time was greater in group 1 (8.0 ± 1.8 months), with a difference of 1.4 months than in group 2 (6.6 ± 2.1 months) (P = 0.042). The measurements of lingual bone thickness at the alveolar crest (C) showed significant differences between the 2 groups (P = 0.025). No significant differences were found in other treatment duration or measurements of root development and alveolar bone mass between the 2 groups.Patients with impacted immature incisors with dilacerated roots were younger at the beginning of the closed-eruption treatment and had a longer traction time than those with impacted immature incisors having straight roots. The root dilaceration had little or no effect on root development and alveolar bone mass after the closed-eruption treatment. The closed-eruption treatment of impacted immature incisors with root dilaceration is suggested to commence as early as possible.

Background The continuous mutation of severe acute respiratory syndrome coronavirus 2 has made the coronavirus disease 2019 (COVID-19) pandemic complicated to predict and posed a severe challenge to the Beijing 2022 Winter Olympics and Winter Paralympics held in February and March 2022. Methods During the preparations for the Beijing 2022 Winter Olympics, we established a dynamic model with pulse detection and isolation effect to evaluate the effect of epidemic prevention and control measures such as entry policies, contact reduction, nucleic acid testing, tracking, isolation, and health monitoring in a closed-loop management environment, by simulating the transmission dynamics in assumed scenarios. We also compared the importance of each parameter in the combination of intervention measures through sensitivity analysis. Results At the assumed baseline levels, the peak of the epidemic reached on the 57th day. During the simulation period (100 days), 13,382 people infected COVID-19. The mean and peak values of hospitalized cases were 2650 and 6746, respectively. The simulation and sensitivity analysis showed that: (1) the most important measures to stop COVID-19 transmission during the event were daily nucleic acid testing, reducing contact among people, and daily health monitoring, with cumulative infections at 0.04%, 0.14%, and 14.92% of baseline levels, respectively (2) strictly implementing the entry policy and reducing the number of cases entering the closed-loop system could delay the peak of the epidemic by 9 days and provide time for medical resources to be mobilized; (3) the risk of environmental transmission was low. Conclusions Comprehensive measures under certain scenarios such as reducing contact, nucleic acid testing, health monitoring, and timely tracking and isolation could effectively prevent virus transmission. Our research results provided an important reference for formulating prevention and control measures during the Winter Olympics, and no epidemic spread in the closed-loop during the games indirectly proved the rationality of our research results. Graphical Abstract

Objectives To identify the genetic cause of one Chinese family with hypoplastic amelogenesis imperfecta (AI) and explore the relationship between genotype and its phenotype. Material and methods One Chinese family with generalized hypoplastic AI was recruited. One deciduous tooth from the proband was subjected to scanning electron microscopy. Whole-exome sequencing was performed and identified mutation was confirmed by Sanger sequencing. Bioinformatics studies were further conducted to analyze potential deleterious effects of the mutation. Results The proband presented a typical hypoplastic AI phenotype whose teeth in deciduous and permanent dentitions showed thin, yellow, and hard enamel surface. The affected enamel in deciduous tooth showed irregular, broken, and collapsing enamel rods with borders of the enamel prisms undulated and structural shapes of prisms irregular. A novel homozygous nonsense mutation in the last exon of the enamelin (ENAM) gene (NM_031889.3; c.2078C>G) was identified in the proband, which was predicted to produce a highly truncated protein (NP_114095.2; p.(Ser693*)). This mutation was also identified in the proband's parents in heterozygous form. Surprisingly, the clinical phenotype of the heterozygous parents varied from a lack of penetrance to mild enamel defects. Additional bioinformatics studies demonstrated that the detected mutation could change the 3D structure of the ENAM protein and severely damaged the function of ENAM. Conclusion The novel homozygous ENAM mutation resulted in hypoplastic AI in the present study. Our results provide new genetic evidence that mutations involved in ENAM contribute to hypoplastic AI.

Objectives The reconstruction of bone defects remains a major clinical issue. Our study aims to investigate the ability of RATEA16 (RA, [CH3CONH] RADARADARADARADA-[CONH2]) for the sustained delivering VEGF and BMP-2 to promote angiogenesis and osteogenesis in bone reconstruction. Materials and methods We prepared and investigated the characterization of RATEA16. The survival of human umbilical vein endothelial cells (HUVECs) and human stem cells of the apical papilla (SCAPs) encapsulated in RATEA16 hydrogel were detected. Then we established RA-VEGF/BMP-2 drug delivery systems and measured their drug release pattern. The effects of RA-VEGF scaffolds on HUVECs angiogenesis were investigated in vitro. Then osteoblastic differentiation capacity of SCAPs with RA-BMP-2 scaffolds was analyzed by ALP activity and expression of osteoblast-related genes. Results A porous nanofiber microstructure endowed this scaffold with the ability to maintain the survival of HUVECs and SCAPs. The RA-VEGF/BMP-2 drug delivery systems exhibited several advantages in vitro: injectability, biodegradability, good biocompatibility and noncytotoxicity. Released rhVEGF165 /BMP-2 were proved to promote angiogenesis of HUVECs as well as osteogenesis of SCAPs abilities. Conclusion RATEA16 loading with VEGF and BMP-2 might be a potential clinical strategy for tissue engineering, especially in bone reconstruction, due to its ability of delivering growth factors effectively and efficiently.

Management of dental caries in adolescents presents a population health challenge; thus, it is important to use national epidemiological data to inform policy and action to improve oral health and address inequalities. The aims of this research were to examine dental caries clusters among 15-year-olds, taking account of caries thresholds, and explore associated factors to inform public health action. Secondary analysis of the oral health data on 2,160 15-year-olds from the 2013 Children’s Dental Health Survey in England, Wales, and Northern Ireland was performed. Hierarchical cluster analysis of dental caries experience was conducted across all surfaces and at 4 decay diagnostic thresholds (clinical: International Caries Detection and Assessment System [ICDAS] 1–6, cavitated: ICDAS 3–6, obvious: ICDAS 4–6, and extensive obvious: ICDAS 5–6 decay). Ordered logistic regression was used to estimate the association of behavioural and psychosocial factors with the clusters generated in relation to both clinical and obvious decay experience which are of clinical and epidemiological relevance. A 4-cluster decay pattern representing “low” to “extremely high” decay experience was observed under each of the dental caries diagnostic criteria. For clinical decay, which includes visual enamel caries, 28.70% had low, 39.77% medium, 26.71% high, and 4.81% extremely high caries status. In the adjusted model, significant risk factors for clinical decay included non-modifiable (sex, region, school type, and area deprivation) and modifiable (higher sugar intake at 4 or more times per day and suboptimal dental attendance) factors. This study suggests 4 distinct dental caries patterns among adolescent children nationally. Dental caries clusters demonstrate the importance of embracing proportionate universalism in addressing dental caries in the population oral health strategy.

Background Dental caries is the most prevalent condition globally. Despite improvements over the past few decades, there remains a significant disease burden in childhood. Epidemiological surveys provide insight to disease patterns and trends, and have traditionally focused on obvious decay which are inconsistent with contemporary clinical criteria. This study examined the distribution of dental caries in 12- and 15-year-olds in England, Wales and Northern Ireland, by severity threshold, at surface, tooth and child level and explored its association with socioeconomic, psychological and behavioural factors. Methods Data from 12- and 15-year-olds in the 2013 Children’s Dental Health Survey (CDHS 2013) were analysed at three levels, taking account of dental caries thresholds which involved recording both clinical decay [visual enamel caries (AV) and above] and obvious decay [non-cavitated dentine lesions (2V) and above]. Negative binomial regression was used to identify factors associated with dental caries experience at both thresholds. Results The prevalence and severity of dental caries experience was higher among 15-year-olds at all levels. Visual change in enamel (AV) was by far the most common stage of caries recorded in both ages. The average number of surfaces with obvious decay experience, which has been the traditional epidemiological threshold, in 12- and 15-year-olds was 2.3 and 3.9 respectively. The corresponding values under the clinical decay threshold were higher, at 3.9 and 5.9 respectively. Visualisation of the distribution of dental caries at surface/tooth-level exhibited horizontal symmetry and to a lesser extent vertical symetry. In the adjusted models for both ages, country/region, school type, area deprivation, high frequency sugar consumption and irregular dental attendance were associated with greater caries experience in both groups. Dental anxiety was inversely associated with caries experience among 15-year-olds. Conclusion This research highlights the importance of recognising dental caries patterns by surface, tooth and child-level amongst adolescents and the value of reporting dental caries distribution by threshold in epidemiological surveys, including its relevance for clinical care. Inclusion of enamel caries reveals the extent of caries management required at a point when non-invasive care is possible, emphasising the importance of prevention through contemporary primary care, which includes supporting self-care.

Autosomal-dominant hypocalcification amelogenesis imperfecta (ADHCAI) is characterized by soft enamel that easily disintegrates and exposed dark dentin. ADHCAI is caused by mutations in a gene called family with sequence similarity 83 member H (FAM83H). To investigate the molecular genetics of ADHCAI, a Chinese family in which three generations exhibited ADHCAI was recruited. The enamel ultrastructure was analysed by environmental scanning electron microscopy (ESEM), which showed altered enamel rod (prism) structures in ADHCAI patients compared to the structures in healthy controls. Mutational analysis of the FAM83H gene identified a novel nonsense mutation (c.1222A>T) in the affected family members that encodes a stop codon at amino acid position 408, causing premature protein truncation (p. K408X). Green fluorescent protein (GFP) and FAM83H fusion protein analyses in vitro showed that normal cytoplasmic accumulation of the FAM83H protein was prevented by the K408X mutation in both rat dental epithelial SF2 cells and human embryonic kidney 293T cells. The mutant fusion protein localized primarily to the nucleus, in contrast to the cytoplasmic subcellular localization of the wild-type FAM83H protein. Our results provide new genetic evidence that mutations in FAM83H contribute to ADHCAI.

Disturbed permanent tooth eruption is common in cleidocranial dysplasia (CCD), a skeletal disorder caused by heterozygous mutation of RUNX2, but the mechanism underlying is still unclear. As it is well known that dental follicle cells (DFCs) play a critical role in tooth eruption, the changed biological characteristics of DFCs might give rise to disturbance of permanent tooth eruption in CCD patients. Thus, primary DFCs from one CCD patient and normal controls were collected to investigate the effect of RUNX2 mutation on the bone remodeling activity of DFCs and explore the mechanism of impaired permanent tooth eruption in this disease. Conservation and secondary structure analysis revealed that the RUNX2 mutation (c.514delT, p.172fs) found in the present CCD patient was located in the highly conserved RUNT domain and converted the structure of RUNX2. After osteogenic induction, we found that the mineralised capacity of DFCs and the expression of osteoblast-related genes, including RUNX2, ALP, OSX, OCN and Col Iα1, in DFCs was severely interfered by the RUNX2 mutation found in CCD patients. To investigate whether the osteogenic deficiency of DFCs from the CCD patient can be rescued by RUNX2 restoration, we performed 'rescue' experiments. Surprisingly, the osteogenic deficiency and the abnormal expression of osteoblast-associated genes in DFCs from the CCD patient were almost rescued by overexpression of wild-type RUNX2 using lentivirus. All these findings indicate that RUNX2 mutation can reduce the osteogenic capacity of DFCs through inhibiting osteoblast-associated genes, thereby disturbing alveolar bone formation, which serves as a motive force for tooth eruption. This effect may provide valuable explanations and implications for the mechanism of delayed permanent tooth eruption in CCD patients.

RUNX2 is an important osteo-specific factor with crucial functions in bone formation and remodelling as well as resorption of teeth. Heterozygous mutation of RUNX2 can cause cleidocranial dysplasia (CCD), a systemic disease with extensive skeletal dysplasia and abnormality of tooth growth. In our study, dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) were isolated, cultured and identified from one patient with CCD and compared with normal controls. This CCD patient was confirmed to have a heterozygous frameshift mutation of RUNX2 (c.514delT, p.Ser172fs) in the previous study. The results showed that the proliferation abilities of DFCs and PDLCs were both disturbed by the RUNX2 mutation in the CCD patient compared with the normal control. A co-culture system of these cells with human peripheral blood mononuclear cells was then used to investigate the effect of RUNX2 mutation on osteoclastogenesis. We found that the RUNX2 mutation in CCD reduced the expression of osteoclast-related genes, such as RUNX2, CTR, CTSK, RANKL and OPG The ability of osteoclastogenesis in DFCs and PDLCs detected by tartrate-resistant acid phosphatase staining in the co-culture system was also reduced by the RUNX2 mutation compared with the normal control. These outcomes indicate that the RUNX2 mutation disturbs the modulatory effects of DFCs and PDLCs on the differentiation of osteoclasts and osteoblasts, thereby interfering with bone remodelling. These effects may contribute in part to the pathological manifestations of retention of primary teeth and delayed eruption of permanent teeth in patients with CCD.

Objectives To explore the role of dental follicle cells (DFCs) with a novel cleidocranial dysplasia (CCD) causative gene RUNX2 mutation (DFCsRUNX2+/m ) in delayed permanent tooth eruption. Materials and methods A CCD patient with typical clinical features was involved in this study. DFCsRUNX2+/m were cultured and DNA was extracted for RUNX2 mutation screening. Measurements of cell proliferation, alkaline phosphatase (ALP) activity, alizarin red staining and osteoblast-specific genes expression were performed to assess osteogenesis of DFCsRUNX2+/m . Co-culture of DFCs and peripheral blood mononuclear cells (PBMCs), followed tartrate-resistant acid phosphatase (TRAP) staining, real-time PCR and western blot were performed to evaluate osteoclast-inductive capacity of DFCsRUNX2+/m . Results A missense RUNX2 mutation (c. 557G>C) was found in DFCsRUNX2+/m from the CCD patient. Compared with normal controls, this mutation did not affect the proliferation of DFCsRUNX2+/m , but down-regulated the expression of osteogenesis-related genes, leading to a decrease in ALP activity and mineralisation. Co-culture results showed that DFCsRUNX2+/m reduced the formation of TRAP+ multinucleated cells and the expression of osteoclastogenesis-associated genes. Furthermore, the mutation reduced the ratio of RANKL/OPG in DFCsRUNX2+/m . Conclusions DFCsRUNX2+/m disturbs bone remodelling activity during tooth eruption through RANK/RANKL/OPG signalling pathway and may thus be responsible for impaired permanent tooth eruption in CCD patients.

Objectives Cleidocranial dysplasia (CCD), mainly caused by RUNX2 mutation, is a dominantly inherited skeletal disorder with many dental abnormalities, characterized by delayed permanent tooth eruption. In this study, we explored a novel RUNX2 mutation and the effect of RUNX2 mutation on osteogenic differentiation of dental follicle cells (DFCs). Design A CCD patient with typical clinical features was involved in this study. Conservation and secondary structural analysis of the RUNX2 mutation was first performed. Then DFCs that stably expressing wild-type or mutant RUNX2 were established using lentiviruses. Cell Counting Kit 8 (CCK8) assays were performed to test the proliferation of DFCs. Measurement of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining and determination of osteoblast-specific genes expression were performed to assess osteogenic capacity of DFCs. Results A missense mutation (c.674 G > T, p. R225 L) of RUNX2 gene was identified in the CCD patient. Conservation and secondary structural analysis revealed that the mutation was located in highly conserved Runt domain and altered secondary structure of RUNX2. CCK8 assays showed that mutant RUNX2 increased the proliferation rate of DFCs compared to wild-type RUNX2. ALP activity, ALP staining and alizarin red staining results indicated that mutant RUNX2 decreased the mineralization ability of DFCs. In addition, mutant RUNX2 significantly down-regulated the expression of osteoblast-associated genes. Conclusions RUNX2 mutation can reduce the osteogenic capacity of DFCs by inhibiting osteoblast-associated genes and then affecting bone formation, which participates in bone remodeling during tooth eruption. These effects may be partly responsible for the defects in permanent tooth eruption of CCD patients.

Cleidocranial dysplasia (CCD) is an autosomal dominant inheritable skeletal disorder characterized by cranial dysplasia, clavicle hypoplasia and dental abnormalities. This disease is mainly caused by heterozygous mutations in RUNX2, a gene that encodes an osteoblast-specific transcription factor. In the present study, mutational analyses of RUNX2 gene were performed on four unrelated Chinese patients with CCD. Four different RUNX2 mutations were detected in these patients, including one nonsense mutation (c.199C>T p.Q67X) and three missense mutations (c.338T>G p.L113R, c.557G>C p.R186T and c.673C>T p.R225W). Among them, two mutations (c.199C>T p.Q67X and c.557G>C p.R186T) were novel and the other two had been reported in previous literatures. Except for Q67X mutation located in the Q/A domain, other three mutations were clustered within the highly conserved Runt domain. Green fluorescent protein (GFP) and RUNX2 fusion protein analyses in vitro showed that nuclear accumulation of RUNX2 protein was disturbed by Q67X mutation, while the other two mutations (c.338T>G p.L113R and c.557G>C p.R186T) had no effects on the subcellular distribution of RUNX2. Luciferase reporter assay demonstrated that all the three novel RUNX2 mutations significantly reduced the transactivation activity of RUNX2 on osteocalcin promoter. Our findings enrich the evidence of molecular genetics that the mutations of RUNX2 gene are responsible for CCD.

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

Background Severe early childhood caries (s-ECC), which has quite high prevalence among children, is a widespread problem with significant impacts among both developing and developed countries. At present, it is widely known that no early detective techniques and diagnostic tests could have high sensitivity and specificity when using for clinical screening of s-ECC. In this study, we had applied magnetic bead (MB)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen distinctive candidate biomarkers of this disease, so as to establish protein profiles and diagnostic models of s-ECC. Methods Firstly, we used the technique mentioned above to detect specifically expressed peptides in saliva samples from ten children with s-ECC, separately at the time point of before, 1 and 4 weeks after dental treatment. Then a diagnostic model for s-ECC was established with the K nearest-neighbour method, which was validated in another six children in the next stage of study. After that, linear ion trap-orbitrap-mass spectrometry (LTQ-Orbitrap-MS) was performed to identify which of the proteins in saliva might be the origination of these peptides. Results We found that seven peptide peaks were significantly different when comparing the three time points, among them two were higher, while other five were lower in the pre-treatment s-ECC group compared with post-treatment. The sensitivity and specificity of the diagnostic model we built were both 83.3 %. Two of these peptides were identified to be segments of histatin-1, which was one important secretory protein in saliva. Conclusions Hereby we confirmed that MB-based MALDI-TOF MS is an effective method for screening distinctive peptides from the saliva of junior patients with s-ECC, and histatin-1 may probably be one important candidate biomarker of this common dental disease. These findings might have bright prospect in future in establishing new diagnostic methods for s-ECC. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0996-4) contains supplementary material, which is available to authorized users.

Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of post-transcriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45 alpha mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45 alpha mRNA degradation. Gadd45a functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Heat shock transcription factor 1 (HSF1) monitors the structural integrity of intracellular proteins and its regulation is essential for the health and longevity of eukaryotic organisms. HSF1 also plays a role in the acute inflammatory response in the negative regulation of cytokine gene transcription. Here we show, for the first time, that HSF1 is regulated by the proinflammatory protein kinase MAPKAP kinase 2 (MK2). We have shown that MK2 directly phosphorylates HSF1 and inhibits activity by decreasing its ability to bind the heat shock elements (HSE) found in the promoters of target genes encoding the HSP molecular chaperones and cytokine genes. We show that activation of HSF1 to bind HSE in hsp promoters is inhibited through the phosphorylation of a specific residue, serine 121 by MK2. A potential mechanism for MK2-induced HSF1 inactivation is suggested by the findings that phosphorylation of serine 121 enhances HSF1 binding to HSP90, a major repressor of HSF1. Dephosphorylation of serine 121 in cells exposed to non-steroidal anti-inflammatory drugs leads to HSP90 dissociation from HSF1, which then forms active DNA binding trimers. These experiments indicate a novel mechanism for the regulation of HSF1 by proinflammatory signaling and may permit HSF1 to respond rapidly to extracellular events, permitting optimal physiological regulation.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by chromosome instability and cancer predisposition. At least 11 complementation groups for FA have been identified, and eight FA genes have been cloned. Interestingly, the eight known FA proteins cooperate in a common pathway leading to the interaction of monoubiquitinated FANCD2 and BRCA2 in damaged chromatin. Disruption of this pathway results in the clinical and cellular abnormalities common to all FA subtypes. This review will examine the interaction of the cloned FA proteins with each other and with other DNA damage response proteins (i.e., ATM, ATR, and NBS1). Also, somatic (acquired) disruption of the FA pathway in human tumors appears to account for their chromosome instability and crosslinker hypersensitivity.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2(-/-) cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2(-/-) cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.

Introduction In this study, we evaluated root and alveolar bone development in unilateral osseous impacted immature maxillary central incisors by cone-beam computed tomography before and after closed-eruption treatment, in comparison with naturally erupted contralateral immature maxillary central incisors. Methods and Results The study included 30 patients, 20 boys and 10 girls, with a mean age of 8.44 ± 1.20 years (range, 6.5-11.2 years). After treatment, the root lengths of both the impacted maxillary central incisors (10.66 ± 2.10 mm) and the contralateral maxillary central incisors (11.04 ± 1.76 mm) were significantly greater than their pretreatment values (6.67 ± 1.94 and 9.02 ± 2.13 mm, respectively). The root canal widths of the incisors decreased significantly after treatment. From the posttreatment cone-beam computed tomography images, the ratio of exposed root length to total root length and the thickness of the alveolar bone at 1 mm under the alveolar crest and at the apex were calculated to evaluate alveolar bone development. Impacted immature maxillary central incisors differed significantly from contralateral immature maxillary central incisors in labial exposed root length, labial ratio to total root length, and lingual alveolar crest. Clinical crown height was higher (statistically but not clinically) for the impacted incisors (9.87 mm) than for the contralateral incisors (9.37 mm). Conclusions Impacted immature incisors grew to the same stage as did erupted contralateral incisors after closed-eruption treatment. Both incisor types had some alveolar bone loss, and thin alveolar bone surrounded the roots.

Purpose: To evaluate the prognostic utility of immunohistochemical assessment of key proteins in multiple DNA repair pathways in triple-negative breast cancer (TNBC; estrogen receptor negative, progesterone receptor negative, and HER2/neu negative by immunohistochemistry). Experimental Design: Archived clinically annotated tumor specimens from 112 women with TNBC were immunostained with antibodies against DNA repair proteins and scored using digital image analysis. The cohort was divided into training and test sets for development of a multiantibody model. Scores were combined with clinical data to assess association with outcome. Results: Low XPF (P = 0.005), pMK2 (P = 0.01), MLH; P = 0.002), and FANCD2 (P = 0.001) were each associated with shorter time to recurrence (TTR) in univariate analysis. A 4-antibody model could segregate high-risk and low-risk groups on the basis of TTR in both the training (relative risk [RR] = 3.52; P = 9.05E-07) and test (RR 2.67; P = 0.019) cohorts. Conclusions: DNA repair proteins may be useful as prognostic markers in TNBC. Further study in larger, uniformly treated cohorts with additional clinical parameters is warranted. Clin Cancer Res; 16(23); 5796–804. ©2010 AACR.

The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.

Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. We have examined the interaction of two key molecular components in the response, heat shock transcription factor 1 (HSF1) and extracellular signal regulated protein kinase (ERK). Whereas both HSF1 and ERK are required to protect cells against apoptosis, ERK activation is paradoxically antagonistic to trans-activation of hsp promoters by HSF1 and HSP accumulation during heat shock. We have found that the two pathways interact directly and that heat shock causes the physical association of ERK1 with HSF1, an interaction that promotes the kinase activity of ERK in heat-shocked cells. ERK activation results in the recruitment of the phosphoserine binding protein 14-3-3epsilon in a manner dependent on previous HSF1 phosphorylation by ERK. The effects of 14-3-3epsilon binding on HSF1 were complex, however, depending on extracellular conditions, in that HSF1-14-3-3 binding at 37 degrees C led to the cytoplasmic sequestration and repression of HSF1, whereas heat shock overrode these effects and caused quantitative nuclear localization of HSF1. Although the effects of 14-3-3epsilon binding to HSF1 were overridden acutely by stress, during recovery from heat shock, 14-3-3epsilon association again led to enhanced cytoplasmic localization of HSF1, implicating a role for ERK/14-3-3epsilon in HSF1 deactivation in recovering cells. Association of HSF1 with ERK and 14-3-3epsilon during heat shock may thus modulate the amplitude of the response and lead to efficient termination of HSP expression on resumption of growth conditions.

Heat shock transcription factor 1 (HSF1) is the mammalian regulator of the heat shock response and activates the transcription of heat shock protein (Hsp) molecular chaperone genes (43, 47, 49). Inactivation of the murine hsf1gene has been shown to confer a complex phenotype, indicating an essential function for hsf1 in growth, in development, and in acute response to stress (35). Disruption of hsf1 (i.e., hsf1−/−) in mouse embryonic fibroblasts leads to a profound loss of thermotolerance and markedly increased susceptibility to heat-induced apoptosis (11, 35). hsf1 is required as a maternal factor during the early cleavage stage of development in the −/− mouse embryo (11). hsf1-deficient mice can survive to adulthood but display severe defects in the chorioallantoic placenta that result in increased prenatal lethality (56). In addition, the aging process is associated with degeneration of the heat shock response, and transcriptional activity of HSF1 protein was significantly reduced with age in a cell-free system, as well as in isolated hepatocytes (26). Understanding the processes involved in HSF1 regulation may therefore aid in delineating its role in resistance to stress, development, and aging. Under normal conditions, cellular HSF1 exists in a predominantly transcriptionally repressed state (44, 59). Such HSF1 is monomeric, is constitutively phosphorylated, and lacks the ability to bind the cis-acting heat shock elements (HSEs) located in the promoters of Hsp genes (50, 55). Induction of transcriptional activity by heat shock then results in the conversion of HSF1 from inactive monomer to a DNA-binding trimer (44, 46, 53). Activation of HSF1 is a multistep process, involving trimerization, acquisition of HSE-binding activity, novel phosphorylation, and transactivation of Hsp genes (38, 50, 55). Trimerization of HSF1 is governed by leucine zipper domains in the N terminus and is subject to intramolecular negative regulation by a fourth leucine zipper domain in the C terminus (44). The molecular chaperone Hsp90 functions as the principal cellular repressor of HSF1 in unstressed cells and plays a major role in retaining HSF1 in an inactive state; HSF1 trimerization is accompanied by the sequestration of Hsp90 in protein aggregates and escape from Hsp90-containing HSF1 complexes in response to stress (59). Hsp70, is also important, but not sufficient, to negatively regulate HSF1 activity in the absence of stress (1, 5). HSF1 also appears to have additional layers of regulation in the cell (25, 42). Our studies, as well as the work of others, have demonstrated the hierarchical phosphorylation of human HSF1 within its transcriptional regulatory domain by extracellular signal-regulated kinase 1 (ERK1; on serine 307) and by glycogen synthase kinase 3 (GSK3; on serine 303) (12, 13, 23, 29, 30). The regulatory domain of HSF1 functions as a molecular switch coupling hsp gene transcription to cellular conditions, repressing C-terminal transactivation domains under growth conditions, and under stress conditions causing powerful stimulation of the same activation domains (22, 30, 40). In the present study, we have examined how the regulatory domain responds to extracellular conditions. Since phosphorylation can directly regulate distinct aspects of transcription factor function, including subcellular localization, protein stability, protein-protein interactions, DNA binding, and transactivation, we have examined the role of phosphorylation in the activity of the regulatory domain (14, 54). Our previous studies indicate that the protein kinases ERK1 and GSK3 phosphorylate HSF1 on serine residues within a proline-rich region (RVKEEPPS303PPQS307PRV) of the regulatory domain (12, 13). Many recent studies show that phosphorylation on serine and threonine can be converted into an intracellular signal by association of the phosphorylated domain with regulatory proteins that recognize serine/threonine phosphorylated domains (14). The first such proteins to be identified were the highly conserved 14-3-3 family, which bind to a wide array of cellular proteins largely through recognition of phosphoserine-containing domains (2, 57). At least seven 14-3-3 genes exist in vertebrates, and these give rise to nine protein isoforms (α, β, δ, ɛ, γ, η, σ, τ, and ζ) (3). 14-3-3 proteins are found largely as dimers within the cell and are able to bind either to multiple sites within proteins such as c-Raf1 or to act as a bridge, with one 14-3-3 dimer binding to two different proteins (20). 14-3-3 dimers can thus act as molecular scaffold proteins, bringing together proteins that interact functionally and effecting phosphorylation-dependent cell regulation (20). These highly abundant proteins are thus involved in key cellular processes, such as signal transduction, cell cycle control, and apoptosis (20, 34, 57, 58). In the present study, we investigated the potential role of 14-3-3 in the function of the transcriptional regulatory domain of HSF1. We show for the first time that HSF1 can bind to 14-3-3. In addition, we have demonstrated that extracellular ERK activation by mitogenic stimulation, a process that controls phosphorylation of serines 303 and 307 of HSF1, leads to HSF1 association with 14-4-3 species, including 14-3-3ɛ and 14-3-3ζ. In addition, inhibiting the ERK cascade with the specific MEK1 inhibitor PD98059 in vivo strongly suppresses the binding of HSF1 to 14-3-3ɛ. We next demonstrated the direct in vitro binding of 14-3-3ɛ to synthetic HSF1-derived peptides phosphorylated at serines 303 and 307. Our studies further showed that 14-3-3ɛ binding inhibits both the transcriptional activity and the nuclear accumulation of HSF1 in HeLa cells at 37°C. These effects of 14-3-3ɛ on nuclear exclusion and transactivation of HSF1 required serines 307 and 303 within the regulatory domain. Leptomycin B (LMB), specific inhibitor of nuclear export receptor CRM1, blocked the cytoplasmic localization of HSF1 in 14-3-3ɛ-overexpressing cells. These experiments suggest that 14-3-3ɛ mediates interaction between phospho-S307-S303-HSF1 and CRM1 in either a direct or an indirect manner and thus contributes to cytoplasmic sequestration and transcriptional repression in response to ERK stimulation. Our experiments therefore indicate a pathway whereby HSF1 activity and molecular chaperone expression are regulated at the intracellular level by protein kinases and within larger biological systems through the mediation of extracellular factors.

Heat shock factor-1 (HSF-1) is the regulator of hsp molecular chaperone transcription, although the intracellular mechanisms involved in HSF-1 activation have not been fully elucidated. As HSF1 is activated by heat shock simultaneously with the nuclear translocation of the protein kinase CK2, we have investigated the role of CK2 in HSF1 activation. We demonstrate that HSF-1 is phosphorylated by CK2 on both serine and threonine residues and has characterized a phosphorylation site at threonine 142. Mutation of Thr-142 to alanine (T142A) inhibits trans-activation of the HSP70 gene by HSF1 and in addition inhibits the accumulation of HSF-1 competent to bind heat shock elements in the nucleus. HSF1 activation by heat is correlated with the thermal activation of nuclear CK2 and overexpression of CK2 activates HSF1. Phosphorylation by CK2 on threonine 142 may therefore be an essential step in the thermal activation of latent HSF1 by stresses.

Heat shock transcription factor 1(HSF1) activation is a multistep process. The conversion of a latent cytoplasmic form to a nuclear, DNA binding state appears to be activated by nonsteroidal anti-inflammatory drugs. In previous studies, we showed that HSF 1 is phosphorylated by the protein kinase RSK2 in vitro and that this effect is inhibited by nonsteroidal anti-inflammatory drugs at the concentration that leads to the activation of HSF1 in vivo (Stevenson et al 1999). In the present study, using cells from a patient with Coffin-Lowry syndrome (deficient in RSK2), we demonstrate that RSK2 slightly represses activation of HSF1 in vivo at 37 degrees C. In Coffin-Lowry syndrome cells, HSF1-HSE DNA binding activity after treatment with sodium salicylate was slightly higher than that in untreated cells, indicating that although RSK2 is involved in HSF1 regulation, it is not the unique protein kinase that suppresses HSF1-HSE binding activity at 37 degrees C. However, heat shock treatment resulted in significantly higher HSF1-HSE binding activity in Coffin-Lowry syndrome cells as compared with normal controls, suggesting that RSK2 represses HSF1-HSE binding activity during heat shock.

Background Healthcare providers generate a huge amount of biomedical data stored in either legacy system (paper-based) format or electronic medical records (EMR) around the world, which are collectively referred to as big biomedical data (BBD). To realize the promise of BBD for clinical use and research, it is an essential step to extract key data elements from unstructured medical records into patient-centered electronic health records with computable data elements. Our objective is to introduce a novel solution, known as a double-reading/entry system (DRESS), for extracting clinical data from unstructured medical records (MR) and creating a semi-structured electronic health record database, as well as to demonstrate its reproducibility empirically. Methods Utilizing the modern cloud-based technologies, we have developed a comprehensive system that includes multiple subsystems, from capturing MRs in clinics, to securely transferring MRs, storing and managing cloud-based MRs, to facilitating both machine learning and manual reading, and to performing iterative quality control before committing the semi-structured data into the desired database. To evaluate the reproducibility of extracted medical data elements by DRESS, we conduct a blinded reproducibility study, with 100 MRs from patients who have undergone surgical treatment of lung cancer in China. The study uses Kappa statistic to measure concordance of discrete variables, and uses correlation coefficient to measure reproducibility of continuous variables. Results Using the DRESS, we have demonstrated the feasibility of extracting clinical data from unstructured MRs to create semi-structured and patient-centered electronic health record database. The reproducibility study with 100 patient’s MRs has shown an overall high reproducibility of 98 %, and varies across six modules (pathology, Radio/chemo therapy, clinical examination, surgery information, medical image and general patient information). Conclusions DRESS uses a double-reading, double-entry, and an independent adjudication, to manually curate structured data elements from unstructured clinical data. Further, through distributed computing strategies, DRESS protects data privacy by dividing MR data into de-identified modules. Finally, through internet-based computing cloud, DRESS enables many data specialists to work in a virtual environment to achieve the necessary scale of processing thousands MRs within days. This hybrid system represents probably a workable solution to solve the big medical data challenge. Electronic supplementary material The online version of this article (doi:10.1186/s12911-016-0357-5) contains supplementary material, which is available to authorized users.