bluuebunny/crossref_metadata_2025 huggingface dataset:
Epilepsy control following intracranial monitoring without resection in young children
Authors: Roth Jonathan , Olasunkanmi Adeolu , Ma Tracy S. , Carlson Chad , Devinsky Orrin , Harter David H. , Weiner Howard L.
Published: January 2012

Purpose: Intracranial monitoring (IM) is a key diagnostic procedure for select patients with treatment‐resistant epilepsy (TRE). Seizure focus resection may improve seizure control in both lesional and nonlesional TRE. IM itself is not considered to have therapeutic potential. We describe a cohort of patients with improved seizure control following IM without resective surgery.

Methods: Over 12.5 years, 161 children underwent 496 surgeries including intracranial monitoring. We retrospectively reviewed the patients’ charts, operative reports, and radiologic scans, under an institutional review board–approved protocol.

Key Findings: Seventeen patients underwent only IM, without additional resective surgery, and seven had a dramatic improvement in their epilepsy; six of the seven patients are seizure‐free (Engel class I), and one rarely has seizures (Engel class II). All seven patients had frequent seizures that led to IM: either daily (five patients) or 1–2 per week (two patients). The mean age (± standard deviation, SD) at seizure onset was 1.6 ± 1.3 years (range 0.5–4 years). Etiologies were tuberous sclerosis (3 patients), trauma (1 patient), and unknown (3 patients). Mean age at surgery (± SD) was 4.1 ± 2 years (range 1–7 years), and duration of epilepsy 2.5 ± 1.1 years (range 0.5–4 years). Duration of IM was 11.7 ± 5.6 days (5–19 days). Six patients had bilateral and one unilateral invasive electrodes. At last follow‐up, four patients required fewer antiepileptic drugs (AEDs), one had the same medication but a higher dose, and two patients were taking additional AEDs. Follow‐up was 30.6 ± 9.5 months (range 19–41 months).

Significance: Although uncommon, patients with TRE may improve after IM alone. The explanation for this observation remains unclear; however, perioperative medications including steroids, direct cortical manipulation, or other factors may influence the epileptogenic network.
The new ILAE report on terminology and concepts for the organization of epilepsies: Critical review and contribution
Authors: Panayiotopoulos Chrysostomos P.
Published: January 2012

This critical review refers to the new report on terminology and concepts for the organization of epilepsies by the Commission of the International League Against Epilepsy (ILAE). It is unfortunate that most of the proposals in the Commission’s report are modified interpretations and nomenclature of previous ILAE classifications; new terms are not better than the old ones, and recent advances have not been incorporated. Hence, the new ILAE report met with considerable dissatisfaction from several expert epileptologists. The Commission abandoned (1) the disease‐syndrome distinction, although “disease” is generally differentiated from “syndrome” in most medical texts as well as in the ILAE classification itself; (2) the distinction of “generalized” and “focal” for epileptic syndromes, despite maintaining this distinction for epileptic seizures and despite the fact that most epileptic syndromes manifest exclusively either with generalized or focal epileptic seizures; (4) the terms “idiopathic,”“symptomatic,” and “cryptogenic,” although these terms have been well defined in the previous ILAE classifications; reiterating their true meaning would be sufficient. Genetic epilepsy could be a new category and (5) the designation of “benign” epilepsies, despite the recommendations of experts at the Monreale workshop. In addition, the Commission proposed that “age at onset” be used as a primary dimension for organizing the epilepsies. However, (a) this runs counter to classification efforts of other diseases in medicine and neurology; (b) syndromes that are likely to be linked together on the basis of electroclinical (and often genetic) evidence are now separated and intermixed with a number of heterogeneous epilepsies; and (c) a considerable number of epileptic syndromes have a wide range of age at onset from childhood to adulthood. Furthermore, epilepsy syndromes were given by name only, without definition; thus we remain dependent on previous ILAE definitions, which are often broad and imprecise. The ILAE should commission consensus of opinion from experts in specific fields in order to define each syndrome. Areas of certainties and uncertainties and of agreements and disagreements should be identified and explained. This approach may be the only way toward achieving a scientifically sound and clinically meaningful organizational system for the epileptic seizures and the epilepsies—a process that would incorporate the tremendous advances in our field and would be accepted by the wider community of clinicians and scientists.
Placebo‐corrected efficacy of modern nonenzyme‐inducing AEDs for refractory focal epilepsy: Systematic review and meta‐analysis
Authors: Beyenburg Stefan , Stavem Knut , Schmidt Dieter
Published: January 2012

Purpose:  Given serious concerns over the adverse effects of enzyme induction, modern nonenzyme‐inducing antiepileptic drugs (AEDs) may be preferable, provided they have similar efficacy as enzyme‐inducing AEDs. This is currently unclear.

Methods:  Therefore, we performed a meta‐analysis of the evidence to determine the placebo‐corrected efficacy of adjunctive treatment with modern nonenzyme‐inducing AEDs versus modern enzyme‐inducing AEDs that are on the market for refractory focal epilepsy.

Key Findings:  Of 322 potentially eligible articles reviewed in full text, 129 (40%) fulfilled eligibility criteria. After excluding 92 publications, 37 studies dealing with a total of 9,860 patients with refractory focal epilepsy form the basis for the evidence. The overall weighted pooled‐risk ratio (RR) in favor of enzyme‐inducing AEDs over placebo was 2.37 (95% confidence interval [CI] 1.77–3.18, p < 0.001) for at least 50% seizure reduction and 4.45 (2.26–8.76, p < 0.001) for seizure freedom. The corresponding weighted pooled RR in favor of nonenzyme‐inducing AEDs over placebo was 2.28 (95% CI 2.03–2.57, p < 0.001) for at least 50% seizure reduction and 3.23 (95% CI 2.23–4.67, p < 0.001) for seizure freedom. In a meta‐regression analysis in the same sample with at least 50% seizure reduction as outcome, the ratio of RRs for enzyme‐inducing AEDs (eight studies) versus nonenzyme‐inducing AEDs (29 studies) was 1.01 (95% CI 0.77–1.34, p = 0.92)). Similarly, the ratio of RRs for a seizure‐free outcome for enzyme‐inducing AEDs (six studies) versus nonenzyme‐inducing AEDs (19 studies) was 1.38 (95% CI 0.60–3.16, p = 0.43).

Significance:  Although the presence of moderate heterogeneity may reduce the validity of the results and limit generalizations from the findings, we conclude that the efficacy of adjunctive treatment with modern nonenzyme‐inducing AEDs is similar to that of enzyme‐inducing AEDs. Given the negative consequences of enzyme induction, our data suggest that nonenzyme‐inducing AEDs may be useful alternatives to enzyme‐inducing AEDs for treatment of refractory focal epilepsy.
Seasonal association of thrombotic thrombocytopenic purpura
Authors: Park Yara A. , Poisson Jessica L. , McBee Matthew T. , Afenyi‐Annan Araba
Published: January 2012

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a thrombotic microangiopathy, is a clinical diagnosis, characterized by microangiopathic hemolytic anemia and thrombocytopenia without another likely explanation. Some initiators of the disease are well represented in the literature, such as certain drugs, malignancies, and viral illness; however, there are less objective factors still being investigated, with references to hormonal, stress, and seasonal variations considered anecdotally. A better insight of these factors would aid in understanding the pathophysiology of the disease.

STUDY DESIGN AND METHODS: We performed a retrospective review of all idiopathic TTP cases treated with therapeutic plasma exchange at our institution from 1999 to 2008 to determine whether there was seasonal variation in TTP presentation. Seasons were defined as follows: winter = December to February; spring = March to May; summer = June to August; and fall = September to November. With the use of Poisson regression models, the incidence between seasons was compared.

RESULTS: During this study period, a total of 97 cases were recorded. Summer had the highest occurrence of TTP (35%). This was significant compared to the fall (p = 0.012) and the winter (p = 0.019). There were more cases in the summer compared to the spring, but this was not significant.

CONCLUSION: In our population, there was a significant difference in the number of TTP cases presenting in summer compared to fall and winter. This supports a possible environmental, infectious, or physiologic influence associated with the summer.
Radiofrequency Ablation Does Not Induce Apoptosis in the Rat Myocardium
Authors: DOS SANTOS LUIS F. , ANTONIO EDNEI , SERRA ANDREY , VENTURINI GABRIELA , OKADA MIEKO , ARAÚJO SERGIO , TUCCI PAULO , DE PAOLA ANGELO , FENELON GUILHERME
Published: January 2012

Background:
 The mechanisms implicated in the genesis of delayed radiofrequency (RF) effects remain unclear, but may be related to extension of the lesion beyond the region of coagulative necrosis. The role of apoptosis in this process has not been previously reported. We assessed whether RF promotes apoptosis in the region surrounding acute ablation lesions in a rat model.

Methods:
 Wistar rats (n = 30; weight 300 g) were anesthesized, the chest was opened, and the heart was exposed. A modified unipolar RF ablation (custom catheter 4.5‐mm‐tip diameter, 12 Watts, 10 seconds) was undertaken on the left ventricular anterolateral epicardial surface and the chest was closed. After 2 hours, animals were killed for histological (hematoxylin and eosin, TdT‐mediated dUTP Nick End‐Labeling [TUNEL] assay) and immunohistochemical (anti‐BAD and anti‐caspase 3 antibodies) analysis (n = 18). Additional animals (n = 12) were sacrificed at 2 (n = 3), 24 (n = 3), 48 (n = 3), and 72 hours (n = 3) after ablation exclusively for anti‐BAD Western Blotting analysis.

Results:
 Lesions were characterized by well‐defined regions of coagulative necrosis. In 18/18 (100%) animals, TUNEL assay revealed positive luminescent reaction cells in the region surrounding the lesion, extending up to 2 mm from the border zone. However, microscopic evaluation of the nuclei and immunohistochemical and anti‐BAD Western Blotting analysis were negative in all (100%) rats. Thus, positive TUNEL reaction in the periphery of the ablation lesion likely reflects nonspecific DNA damage.

Conclusion:
 RF ablation does not promote apoptosis in the periphery of the myocardial lesion. This finding may have implications for the elucidation of late lesion extension following RF ablation. PACE 2012; 35:449–455)
Transcranial Doppler and Cerebral Augmentation in Acute Ischemic Stroke
Authors: Saqqur Maher , Ibrahim Mohamed , Butcher Ken , Khan Khurshid , Emery Derek , Manawadu Dulka , Derksen Carol , Schwindt Brenda , Shuaib Ashfaq
Published: January 2012

BACKGROUND

Collateral flow augmentation using partial aortic occlusion may improve cerebral perfusion in acute stroke. We assessed the effect of partial aortic occlusion on arterial flow velocities of acute stroke patients.

METHODS

Patients with neurological deficits following thrombolysis were treated with partial aortic occlusion. Transcranial Doppler ultrasound (TCD) was used to measure arterial flow velocities at baseline, before and during balloon inflation. The augmented mean flow velocity (MFV), peak systolic velocity (PSV), and end diastolic velocity flow percentages (aMFV%, aPSV%, aEDV%) were calculated and compared based on outcome.

RESULTS

Of 11 patients, 3 did not have a temporal window and thus were excluded from our analysis. Six of the remaining 8 patients had middle cerebral artery (MCA) occlusions; the final 2 had terminal internal carotid artery (TICA) occlusions. Three of these 8 patients had good outcome at 90 days (mRS < 3).

Before intra‐aortic balloon inflation (IABI), the mean affected artery MFV was 23 ± 11 cm/s; during the procedure it was 26 ± 12 cm/s (P = .2). Mean affected artery PSV at baseline and during balloon inflation were 37 ± 16 and 46 ± 23, respectively (P = .1). Mean augmented affected artery MFV% in patients with good long‐term outcome was 65.4 ± 46, while the result in those with poor outcome was −3.7 ± 21 (P = .03). Three patients developed anterior cross‐filling, and of these 2 had good long‐term outcome.

CONCLUSION

TCD monitoring of patients treated with IABI may help in predicting outcome in this novel device.
Target gene analyses of 39 amelogenesis imperfecta kindreds
Authors: Chan Hui‐Chen , Estrella Ninna M. R. P. , Milkovich Rachel N. , Kim Jung‐Wook , Simmer James P. , Hu Jan C‐C.
Published: January 2012

Chan H‐C, Estrella NMRP, Milkovich RN, Kim J‐W, Simmer JP, Hu JC‐C. Target gene analyses of 39 amelogenesis imperfecta kindreds.  Eur J Oral Sci 2011; 119 (Suppl. 1): 311–323. © 2011 Eur J Oral Sci

Previously, mutational analyses identified six disease‐causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein‐related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X‐linked AI families (100%) had disease‐causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X‐linked AI. Eighteen families showed an autosomal‐dominant pattern of inheritance. Disease‐causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding‐region or splice‐junction mutations were identified in three probands with autosomal‐dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal‐recessive pattern of inheritance, and disease‐causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease‐causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease‐causing mutation in a given kindred.
Kallikrein‐related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel
Authors: Hu Yuanyuan , Hu Jan C‐C. , Smith Charles E. , Bartlett John D. , Simmer James P.
Published: January 2012

Hu Y, Hu JC‐C, Smith CE, Bartlett JD, Simmer JP. Kallikrein‐related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 217–225. © 2011 Eur J Oral Sci

The crowns of matrix metalloproteinase 20 (Mmp20) null mice fracture at the dentino–enamel junction (DEJ), whereas the crowns of kallikrein‐related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9‐wk‐old wild‐type, Klk4 null, and Mmp20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild‐type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in Mmp20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the Mmp20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid‐maturation wild‐type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in Mmp20 null mice because of hypomineralization at the DEJ.
Cell proliferation and apoptosis in enamelin null mice
Authors: Hu Jan C‐C. , Lertlam Rangsiyakorn , Richardson Amelia S. , Smith Charles E. , McKee Marc D. , Simmer James P.
Published: January 2012

Hu JC‐C, Lertlam R, Richardson AS, Smith CE, McKee MD, Simmer JP. Cell proliferation and apoptosis in enamelin null mice.  Eur J Oral Sci 2011; 119 (Suppl. 1): 329–337. © 2011 Eur J Oral Sci

Enamelin is a secreted glycoprotein that is critical for dental enamel formation. Ameloblasts in enamelin (Enam) null mice develop atypical features that include the absence of a Tomes’ process, expanded endoplasmic reticulum, apparent loss of polarity, and pooling of extracellular matrix in all directions, including between ameloblasts and the stratum intermedium. We hypothesized that ameloblast pathological changes may be associated with increased cell apoptosis. Our objective was to assess apoptotic activity in maxillary first molars of wild‐type, Enam+/−, and Enam−/− mice at postnatal days 5, 7, 9, 14, and 17. Mouse maxillae were characterized by light microscopy after terminal deoxynucleotidyl transferase (TdT)‐mediated biotin–dUTP nick‐end labelling (TUNEL) or 5‐bromo‐2′‐deoxyuridine (BrdU) staining. Following the initial deposition of dentin matrix, ameloblasts became highly dysplastic and no enamel crystal ribbons were deposited. Ameloblast apoptosis was observed in the Enam null mice starting in the secretory stage and with no apparent alteration in cell proliferation. We conclude that in the absence of enamelin and subsequent shutdown of enamel formation, ameloblasts undergo pathological changes early in the secretory stage that are evident as radically altered cell morphology, detachment from the tooth surface, apoptosis, and formation of ectopic calcifications both outside and inside the dystrophic enamel organ.
Biphasic influence of hypoxia on tuftelin expression in mouse mesenchymal C3H10T1/2 stem cells
Authors: Deutsch Dan , Silverstein Nechama , Shilo Dekel , Lecht Shimon , Lazarovici Philip , Blumenfeld Anat
Published: January 2012

Deutsch D, Silverstein N, Shilo D, Lecht S, Lazarovici P, Blumenfeld A. Biphasic influence of hypoxia on tuftelin expression in mouse mesenchymal C3H10T1/2 stem cells.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 55–61. © 2011 Eur J Oral Sci

Tuftelin, an acidic protein, thought to play a role in the initial stages of ectodermal enamel mineralization, has since been detected in mesenchymal‐derived tissues. During bone/cartilage development and regeneration, mesenchymal stem cells (MSCs) undergo an avascular period in a hypoxic environment, involving induction of hypoxia‐inducible factor 1‐alpha (HIF‐1‐alpha), a key component in this process. In the present study we investigated, in a mouse mesenchymal C3H10T1/2 stem cell model, the hypothesis that oxygen stress modulates tuftelin 1 expression in relation to HIF‐1‐alpha (Hif1a), in a mouse mesenchymal C3H10T1/2 stem cell model. The results of the present study showed a biphasic induction of tuftelin, similar to the pattern of HIF‐1‐alpha expression, in MSCs subjected to a hypoxic insult of 1% O2 through a period of 2–24 h. Immunocytochemistry analysis of the cells exposed to hypoxic insult for 2–24 h revealed the same biphasic pattern of tuftelin protein expression. Tuftelin localization appears to be mainly in the cytoplasm, and concentrated at the perinuclear region of the cells by 24 h of hypoxic insult. Based on our previous studies using the neuronal PC12 cell model, in which tuftelin induction was mediated by Hif1a, we propose that tuftelin is a member of oxygen‐sensitive genes and implicated in the adaptive mechanisms regulating MSC function.
Identification of a pH‐responsive DNA region upstream of the transcription start site of human NBCe1‐B
Authors: Snead Christian M. , Smith Susan M. , Sadeghein Negar , Lacruz Rodrigo S. , Hu Ping , Kurtz Ira , Paine Michael L.
Published: January 2012

Snead CM, Smith SM, Sadeghein N, Lacruz RS, Hu P, Kurtz I, Paine ML. Identification of a pH‐responsive DNA region upstream of the transcription start site of human NBCe1‐B.  Eur J Oral Sci 2011; 119 (Suppl. 1): 136–141. © 2011 Eur J Oral Sci

In rodent incisors two distinct stages of enamel formation can be identified visually based on cell morphology: the secretory stage and the maturation stage. The expression profiles of many genes characterize both stages, including the bicarbonate transport protein NBCe1. Bicarbonate is a requirement for the mineralizing enamel matrix to buffer excessive protons that form as a consequence of hydroxyapatite formation. NBCe1‐B mRNA is up‐regulated during the maturation stage of amelogenesis, where hydroxyapatite formation predominates. In this study, a presumed 572‐bp NBCe1‐B promoter region was subcloned into a reporter construct, and within this 572‐bp region of DNA we characterized a 285‐bp segment that shows an increase of ∼ 2.3‐fold in gene‐transcription activity when transfected into ameloblast‐like cells and cultured in medium maintained at pH 6.8 (vs. pH 7.4). A presumed pH‐responsive transcriptional factor‐binding domain(s) thus resides in the 285‐bp NBCe1‐B promoter region where candidate domains include the nuclear factor of kappa light polypeptide gene enhancer in B‐cells1(NFKB1), jun proto‐oncogene (JUN), and tumor protein p53(TP53)‐binding sites. Mutagenesis studies identify that both the NFKB1‐ and TP53‐binding sites are responsive to changes in the extracellular pH. These data help to explain how ameloblasts respond to the altered extracellular milieu of protons by changing their gene‐expression profile throughout the stages of amelogenesis.
Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families
Authors: Wang Shih‐Kai , Chan Hui‐Chen , Rajderkar Sudha , Milkovich Rachel N. , Uston Karen A. , Kim Jung‐Wook , Simmer James P. , Hu Jan C‐C.
Published: January 2012

Wang S‐K, Chan H‐C, Rajderkar S, Milkovich RN, Uston KA, Kim J‐W, Simmer JP, Hu JC‐C. Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families.  Eur J Oral Sci 2011; 119 (Suppl. 1): 158–167. © 2011 Eur J Oral Sci

Dentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD‐II) and dentinogenesis imperfecta types II and III (DGI‐II and DGI‐III, respectively). We identified two kindreds with DGI‐II who exhibited vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and of the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged dentin phosphoprotein (DPP) sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their probable effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice, support the hypothesis that all DSPP mutations cause pathology through dominant‐negative effects. Noting that Dspp is transiently expressed by mouse pre‐ameloblasts during formation of the dentino–enamel junction, we hypothesize that DSPP dominant‐negative effects potentially cause cellular pathology in pre‐ameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.
Gene‐expression analysis of early‐ and late‐maturation‐stage rat enamel organ
Authors: Lacruz Rodrigo S. , Smith Charles E. , Chen Yi‐Bu , Hubbard Michael J. , Hacia Joseph G. , Paine Michael L.
Published: January 2012

Lacruz RS, Smith CE, Chen Y‐B, Hubbard MJ, Hacia JG, Paine ML. Gene‐expression analysis of early‐ and late‐maturation‐stage rat enamel organ  Eur J Oral Sci 2011; 119 (Suppl. 1): 149–157. © 2011 Eur J Oral Sci

Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early‐ and late‐maturation stages of rat incisor and analyzed the global gene‐expression profiles at each stage. Sixty‐three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome‐wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up‐regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down‐regulated during the late maturation stage (n = 25) included those with functions related to cell adhesion, cell signaling, and T‐cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.
Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse
Authors: Zhang Xu , Diekwisch Thomas G. H. , Luan Xianghong
Published: January 2012

Zhang X, Diekwisch TGH, Luan X. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 270–279. © 2011 Eur J Oral Sci

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell–cell and cell–matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26‐amino‐acid deletion in mAMBN, corresponding to a helix‐loop‐helix frameshift. The human AMBN domain (174Q–201G), homologous to the mAMBN 157E–178I helix‐loop‐helix region, formed a helix–loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin‐binding domains, CD63‐interaction domains, and calcium‐binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.
An in vitro scanning microradiography study of the reduction in hydroxyapatite demineralization rate by statherin‐like peptides as a function of increasing N‐terminal length
Authors: Shah Saleha , Kosoric Jelena , Hector Mark P. , Anderson Paul
Published: January 2012

Shah S, Kosoric J, Hector MP, Anderson P. An in vitro scanning microradiography study of the reduction in hydroxyapatite demineralization rate by statherin‐like peptides as a function of increasing N‐terminal length. Eur J Oral Sci 2011; 119 (Suppl. 1): 13–18. © 2011 Eur J Oral Sci

Enamel demineralization is slowed by salivary proteins that inhibit calcium hydroxyapatite (HA) demineralization. Statherin (StN43), a 43‐residue phosphorylated salivary protein with primary sequence similarities to osteopontin and caseins, binds calcium and HA. The aim of this study was to identify the minimum length of the functional domain of the statherin molecule required for cariostatic function by measuring the efficacy of peptides of progressively shorter length (i.e. containing only the N‐terminal 21 (StN21), 15 (StN15), 10 (StN10), or 5 (StN5) residues) to reduce HA demineralization rates (RDHA). Porous HA blocks were used as enamel analogues, and were exposed to 0.1 M acetic acid at pH 4 for 120 h, rinsed, and treated with StN21, StN15, StN10, or StN5 peptides (1.88 × 10−5 M) for 24 h, then demineralized for a further 120 h. The RDHA was measured, before and after peptide treatment, using scanning microradiography. Hydroxyapatite blocks treated with StN21 and StN15 demonstrated a 50–60% reduction in the RDHA. However, no reduction in the RDHA was observed following treatment with either StN10, StN5, or buffer only. The mechanism by which statherin‐like peptides reduce RDHA may be associated with their binding to HA surfaces. Comparisons with previously published binding energies of statherin to HA also suggest that statherin‐like peptides containing 15 N‐terminal residues or more, are required for binding, suggesting a link between binding and demineralization reduction.
Effect of phosphorylation on the interaction of calcium with leucine‐rich amelogenin peptide
Authors: Le Norcy Elvire , Kwak Seo‐Young , Allaire Marc , Fratzl Peter , Yamakoshi Yasuo , Simmer James P. , Margolis Henry C.
Published: January 2012

Le Norcy E, Kwak S‐Y, Allaire M, Fratzl P, Yamakoshi Y, Simmer JP, Margolis HC. Effect of phosphorylation on the interaction of calcium with leucine‐rich amelogenin peptide.  Eur J Oral Sci 2011; 119 (Suppl. 1): 97–102. © 2011 Eur J Oral Sci

Amelogenin undergoes self‐assembly and plays an essential role in guiding enamel mineral formation. The leucine‐rich amelogenin peptide (LRAP) is an alternative splice product of the amelogenin gene and is composed of the N terminus (containing the only phosphate group) and the C terminus of full‐length amelogenin. This study was conducted to investigate further the role of phosphorylation in LRAP self‐assembly in the presence and absence of calcium using small angle X‐ray scattering (SAXS). Consistent with our previous dynamic light‐scattering findings for phosphorylated (+P) and non‐phosphorylated (−P) LRAP, SAXS analyses revealed radii of gyration (Rg) for LRAP(−P) (46.3–48.0 Å) that were larger than those for LRAP(+P) (25.0–27.4 Å) at pH 7.4. However, added calcium (up to 2.5 mM) induced significant increases in the Rg of LRAP(+P) (up to 46.4 Å), while it had relatively little effect on LRAP(−P) particle size. Furthermore, SAXS analyses suggested compact folded structures for LRAP(−P) in the presence and absence of calcium, whereas the conformation of LRAP(+P) changed from an unfolded structure to a more compact structure upon the addition of calcium. We conclude that the single phosphate group in LRAP(+P) induces functionally important conformational changes, suggesting that phosphorylation may also influence amelogenin conformation and protein–mineral interactions during the early stages of amelogenesis.
Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth
Authors: Bronckers Antonius L. J. J. , Guo Jing , Zandieh‐Doulabi Behrouz , Bervoets Theodore J. , Lyaruu Donacian M. , Li Xiangming , Wangemann Philine , DenBesten Pamela
Published: January 2012

Bronckers ALJJ, Guo J, Zandieh‐Doulabi B, Bervoets TJ, Lyaruu DM, Li X, Wangemann P, DenBesten P. Developmental expression of solute carrier family 26A member 4 (SLC26A4/pendrin) during amelogenesis in developing rodent teeth.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 185–192. © 2011 Eur J Oral Sci

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion‐transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT‐PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4‐null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.
Fluoride incorporation into apatite crystals delays amelogenin hydrolysis
Authors: DenBesten Pamela K. , Zhu Li , Li Wu , Tanimoto Kotaro , Liu Haichuan , Witkowska Halina E.
Published: January 2012

DenBesten PK, Zhu L, Li W, Tanimoto K, Liu H, Witkowska HE. Fluoride incorporation into apatite crystals delays amelogenin hydrolysis.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 3–7. © 2011 Eur J Oral Sci

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride‐containing carbonated hydroxyapatite (F‐CAP) synthesized to contain 100, 1,000, or 4,000 ppm F−. After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein‐related peptidase 4 (KLK4), bound protein was characterized by reverse‐phase high‐performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100‐CAP was significantly reduced in a dose‐dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100‐CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100‐CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride‐containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.
Distribution of enamel crystallite orientation through an entire tooth crown studied using synchrotron X‐ray diffraction
Authors: Simmons Lisa M. , Al‐Jawad Maisoon , Kilcoyne Susan H. , Wood David J.
Published: January 2012

Simmons LM, Al‐Jawad M, Kilcoyne SH, Wood DJ. Distribution of enamel crystallite orientation through an entire tooth crown studied using synchrotron X‐ray diffraction.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 19–24. © 2011 Eur J Oral Sci

The biomineralization of human dental enamel has resulted in a highly anisotropic and heterogeneous distribution of hydroxyapatite crystallites, which in combination with its high mineral content has resulted in one of the most durable and hardest tissues in the human body. In this study, we used position‐sensitive synchrotron X‐ray diffraction to quantify the spatial variation in the direction and magnitude of the preferred orientation of enamel crystallites across a whole tooth crown. Two‐dimensional synchrotron X‐ray diffraction images were collected with 300 μm spatial resolution over a series of six sequential tooth sections obtained from a single maxillary first premolar and were analyzed using Rietveld refinement. Both the magnitude and the direction of the crystallite orientation were found to have a high spatial heterogeneity. Areas of high crystallite alignment were directed perpendicular to the biting surfaces, which is thought to meet the functional requirements of mastication. The results may assist in our understanding of the structure–function relationship and of the evolutionary development of enamel.
Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow
Authors: Tamburstuen Margareth V. , Snead Malcolm L. , Reseland Janne E. , Paine Michael L. , Lyngstadaas Staale P.
Published: January 2012

Tamburstuen MV, Snead ML, Reseland JE, Paine ML, Lyngstadaas SP. Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow. Eur J Oral Sci 2011; 119 (Suppl. 1): 286–292. © 2011 Eur J Oral Sci

Ameloblastin (AMBN) was originally described as a tooth‐specific extracellular matrix protein, but current data have shown that AMBN is present in many different tissues of mesenchymal origin. The identification of regulatory elements in the promoter region of theAmbngene would assist in identifying potential mesenchymal‐specific transcriptional factors. In this study we subcloned a 3,788‐bp region upstream (and a 54‐bp region downstream) of the mouseAmbntranscriptional start site into aLacZreporter construct and called this construct 3788‐Ambn‐lacZ.In silicoanalysis of the 3,788‐bpAmbnpromoter region identified 50 potential cis‐regulatory elements, 29 of which are known to be functional in cell populations of mesenchymal origin. The reporter construct was activated in transfected bone marrow cells, and the promoter activity was induced in cell cultures following addition of recombinant AMBN, interferon‐γ, serotonin, or dexamethasone. We discuss the relative significance of the potential cis‐acting gene‐regulatory elements ofAmbnin relation to bone morphogenesis. Knowledge ofAmbngene‐regulatory elements will be of importance when developing strategies for bone repair and replacement in a clinical surgical setting.
Regulation of calcium phosphate formation by amelogenins under physiological conditions
Authors: Kwak Seo‐Young , Green Samantha , Wiedemann‐Bidlack Felicitas B. , Beniash Elia , Yamakoshi Yasuo , Simmer James P. , Margolis Henry C.
Published: January 2012

Kwak S‐Y, Green S, Wiedemann‐Bidlack FB, Beniash E, Yamakoshi Y, Simmer JP, Margolis HC. Regulation of calcium phosphate formation by amelogenins under physiological conditions.  Eur J Oral Sci 2011; 119 (Supp. 1): 103–111. © 2011 Eur J Oral Sci

Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full‐length phosphorylated (P173) and non‐phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C‐terminus. Calcium phosphate formation was assessed by pH change, while the minerals formed were characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Amelogenin self‐assembly was also studied using dynamic light scattering and TEM. The results indicate that IS does not influence the effects of rP147, rP172, and P173 on mineralization. However, in contrast to the findings for low IS, where both P173 and P148 stabilize initially formed amorphous calcium phosphate (ACP) nanoparticles for >1 d, elongated hydroxyapatite crystals were observed after 24 h using P148 at high IS, unlike that seen with P173. Differences in self‐assembly help explain these findings, which suggest that P173 and P148 may play different roles in regulating enamel mineral formation. The present data support the notion that proteolytic processing of P173 is required in vivo to induce the transformation of initial ACP phases to apatitic enamel crystals.
Human osteoblastic cells discriminate between 20‐kDa amelogenin isoforms
Authors: Riksen Elisabeth A. , Petzold Christiane , Brookes Steven , Lyngstadaas Ståle P. , Reseland Janne E.
Published: January 2012

Riksen EA, Petzold C, Brookes S, Lyngstadaas SP, Reseland JE. Human osteoblastic cells discriminate between 20‐kDa amelogenin isoforms.  Eur J Oral Sci 2011; 119 (Suppl. 1): 357–365. © 2011 Eur J Oral Sci

Enamel matrix derivative (EMD) is used to stimulate healing of alveolar bone after destructive marginal periodontitis; however, the roles of the different EMD constituents are unclear. The aim here was to compare the effect of two EMD fractions (A1 and A2) on primary human osteoblasts cultured in the presence of 50 μg ml−1 of A1, A2, or EMD. SDS‐PAGE showed that A1 and A2 were comprised of amelogenins migrating at around 20 kDa. Fourier transform infrared (FTIR) analysis revealed that A1 and A2 had different secondary structures, and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) identified different peptide mass values. Osteoblasts responded differently to A1 and A2. Whereas A1 enhanced the proliferation [measured by the incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU)] of osteoblasts, the expression of runt‐related transcription factor‐2 (RUNX2) mRNA, and the secretion of interleukin 6 (IL‐6) into the cell culture medium, exposure to A2 resulted in increased alkaline phosphatase (ALP) activity, increased expression of CD44 mRNA, and increased secretion of osteoprotegrin (OPG) and receptor activator of nuclear factor‐kappaB ligand (RANKL). The level of osteocalcin in the cell culture medium was increased after all treatments, while A2 stimulated the expression of dentin matrix protein 1 (DMP1) mRNA. The results suggest that both A1 and A2 participate in the observed effect of EMD, but have different effects on the expression of osteoblast mRNA and the secretion of osteoblast protein, and thus might facilitate the differentiation of a different phenotype.
Biophysical characterization of recombinant human ameloblastin
Authors: Wald Tomáš , Bednárová Lucie , Osička Radim , Pachl Petr , Šulc Miroslav , Lyngstadaas Stale Petter , Slaby Ivan , Vondrášek Jiří
Published: January 2012

Wald T, Bednárová L, Osička R, Pachl P, Šulc M, Lyngstadaas SP, Slaby I, Vondrášek J. Biophysical characterization of recombinant human ameloblastin.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 261–269. © 2011 Eur J Oral Sci

Ameloblastin (AMBN) is a protein expressed mainly during dental hard tissue development. Biochemically, it is classified as an intrinsically disordered protein (IDP). Its biological role remains largely unknown; however, the question of AMBN function will undoubtedly be connected to its structural properties and its potential for protein–protein and protein–cell interactions. A basic biophysical characterization of human recombinant ameloblastin (hrAMBN) and its N‐ and C‐terminal domains by means of circular dichroism spectroscopy and dynamic light scattering showed that under physiological conditions ameloblastin is an IDP with a prevalent polyproline‐II (PPII) conformation. Both the N‐ and C‐terminal polypeptides, when expressed independently, showed different structural preferences upon heating as well as different behaviour in the presence of trifluoroethanol and CaCl2 salt. The N‐terminal peptide showed a more ordered structure with a strong tendency to adopt a helical conformation upon the addition of trifluorethanol, whereas the C‐terminal domain seemed to be primarily responsible for the structural disorder of the entire AMBN molecule.
Molecular and circadian controls of ameloblasts
Authors: Athanassiou‐Papaefthymiou Maria , Kim Doohak , Harbron Lindsay , Papagerakis Silvana , Schnell Santiago , Harada Hidemitsu , Papagerakis Petros
Published: January 2012

Athanassiou‐Papaefthymiou M, Kim D, Harbron L, Papagerakis S, Schnell S, Harada H, Papagerakis P. Molecular and circadian controls of ameloblasts.
 
Eur J Oral Sci 2011; 119 (Suppl. 1): 35–40. © 2011 Eur J Oral Sci

Stage‐specific expression of ameloblast‐specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities (i.e. enamel protein secretion and enamel mineralization). This time‐related control is orchestrated by oscillations of clock proteins involved in the regulation of circadian rhythms. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of the transcription factors distal‐less homeobox 3 (Dlx3) and runt‐related transcription factor 2 (Runx2), and the clock gene nuclear receptor subfamily 1, group D, member 1 (Nr1d1), on secretory and maturation ameloblasts [using stage‐specific markers amelogenin (Amelx), enamelin (Enam), and kallikrein‐related peptidase 4 (Klk4)] were evaluated in the HAT‐7 ameloblast cell line. Amelx and Enam steady‐state mRNA expression levels were down‐regulated in Runx2 over‐expressing cells and up‐regulated in Dlx3 over‐expressing cells. In contrast, Klk4 mRNA was up‐regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast‐specific genes but also influenced the expression of Runx2. Multiscale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.
Regulated Exocytosis: Novel Insights from Intravital Microscopy
Authors: Masedunskas Andrius , Porat‐Shliom Natalie , Weigert Roberto
Published: January 2012

Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electrophysiology and electron microscopy. However, recent developments in time‐lapse light microscopy have made possible imaging individual exocytic events, hence, advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy (IVM), a light microscopy‐based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. IVM has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for IVM being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions.