H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Histone H3 tail binds a unique sensing pocket in EZH2 to activate the PRC2 methyltransferase.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 "senses" H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.

S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3.

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

Sinus floor elevation procedures to enable implant placement and integration: techniques, biological aspects and clinical outcomes.

Implant treatment in an atrophied edentulous posterior maxilla constitutes a challenge for the therapeutic team. The authors of the present study acknowledge that modern micro-rough surface implants in lengths of about 8-10 mm or longer and of different brands are similarly successful. Consequently, the authors propose that the use of different sinus floor elevation techniques should be considered when < 8 mm of bone is available below the maxillary sinus. The type of sinus floor elevation technique selected is mainly based on residual vertical bone height, marginal bone width, local intrasinus anatomy and the number of teeth to be replaced, although other factors (such as surgical training and surgical experience) may have an impact. It is proposed that a transcrestal sinus floor elevation approach can be considered as a first-choice method for single tooth gaps in situations with sufficient width for implant placement and a residual bone height of 5-8 mm, while lateral sinus floor elevation, with or without grafting materials, is indicated when < 5 mm of bone is available and when several teeth are to be replaced. With regard to time of implant placement, a one-stage procedure is preferred provided that high primary stability can be ensured.

Neutrophil-like cells derived from the HL-60 cell-line as a genetically-tractable model for neutrophil degranulation.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Alveolar bone remodeling in virtually planned, bone-grafted vs non-grafted guided flapless implant surgery in the anterior maxilla: a cross-sectional retrospective follow-up study.

PURPOSE: In patients who underwent virtual planning and guided flapless implant surgery for teeth missing in the anterior maxilla, we compared buccal bone loss between those treated with and without autogenous bone augmentation. METHODS: Of 22 patients with teeth missing because of trauma or aplasia, 10 (18 implant sites) were reconstructed with buccally placed bone graft harvested from the mandibular ramus, and 12 were non-reconstructed (16 sites). Baseline cone-beam computed tomography allowed for implant planning using the NobelClinician® software and was performed again at 1 year after functional loading. The marginal bone level was assessed radiographically at post-implant baseline and at follow-up. RESULTS: At follow-up, buccal bone loss differed significantly between groups at the central level of the implant (p = 0.0005) but not at the coronal level (p = 0.329). The mean marginal bone level change was 0.6 mm, with no significant between-group difference (p = 0.876). The actual implant position often deviated in the vertical or sagittal plane by an average of 0.3-0.6 mm from the planned position. CONCLUSION: Compared with non-reconstructed patients, reconstructed patients experienced significantly more buccal bone loss at the central level of implants. The groups did not differ at the coronal level or in marginal bone loss, possibly because of the more augmented bone at the central level among reconstructed patients. Differences between planned versus actual implant positions should be considered in situations of limited bone volume at the planned implant site.

Signaling dynamics distinguish high- and low-priority neutrophil chemoattractant receptors.

Human neutrophils respond to multiple chemoattractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils prioritize pathogen- and injury-derived signals over long-range inflammatory signals, such as the leukotriene LTB4, secreted by host cells. Different chemoattractants can also drive qualitatively different modes of migration even though their receptors couple to the same Gαi family of G proteins. Here, we used live-cell imaging to demonstrate that the responses differed in their signaling dynamics. Low-priority chemoattractants caused transient responses, whereas responses to high-priority chemoattractants were sustained. We observed this difference in both primary neutrophils and differentiated HL-60 cells, for downstream signaling mediated by Ca2+, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. The rapid attenuation of Cdc42 activation in response to LTB4 depended on the phosphorylation sites Thr308 and Ser310 in the carboxyl-terminal tail of its receptor LTB4R in a manner independent of endocytosis. Mutation of these residues to alanine impaired chemoattractant prioritization, although it did not affect chemoattractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes between receptors and contributes to chemoattractant prioritization.

Sinus bone formation and implant survival after sinus membrane elevation and implant placement: a 1- to 6-year follow-up study.

OBJECTIVES: To investigate the long-term clinical and radiographic results of the maxillary sinus membrane elevation technique where implants were inserted in a void space created by the elevation of the sinus membrane without adding any graft material. MATERIALS AND METHODS: A total of 84 patients were subjected to 96 membrane elevation procedures and simultaneous placement of 239 implants. Changes of intra-sinus and marginal bone height in relation to the implants were measured in intraoral radiographs taken at insertion, after 6 months of healing, after 6 months of loading and then annually. Computerized tomography was performed pre-surgically and 6 months post-surgically. Resonance Frequency Analyses measurements were performed at the time of implants placement, at abutment connection and after 6 months of loading. The implant follow-up period ranged from a minimum of one to a maximum of 6 years after implants loading. RESULTS: All implants were stable after 6 months of healing. A total of three implants were lost during the follow-up period giving a survival rate of 98.7%. Radiography demonstrated on average 5.3±2.1 mm of intra-sinus new bone formation after 6 months of healing. RFA measurements showed adequate primary stability (implant stability quotient 67.4±6.1) and small changes over time. CONCLUSION: Maxillary sinus membrane elevation and simultaneous placement of implants without the use of bone grafts or bone substitutes result in predictable bone formation with a high implant survival rate of 98.7% during a follow-up period of up to 6 years. The intra-sinus bone formation remained stable in the long-term follow-up. It is suggested that the secluded compartment allowed for bone formation according to the principle of guided tissue regeneration. The high implant survival rate of 98.7% indicated that the implants sufficiently supported the fixed bridges throughout the study period. This technique reduces the risks for morbidity related to harvesting of bone grafts and eliminates the costs of grafting materials.

Clinical outcome and factors determining new bone formation in lateral sinus membrane elevation with simultaneous implant placement without grafting material: A cross-sectional, 3-17 year follow-up study.

BACKGROUND: Lateral sinus membrane elevation with simultaneous implant placement without grafting material (graft-less LSFE) is a widely investigated method for bone augmentation of the maxillary sinus floor. Long-term follow-up studies are rare. PURPOSE: This study aimed to investigate the long-term effects of implants placed with graft-less LSFE. MATERIALS AND METHODS: The study group was comprised of 111 patients previously treated with graft-less LSFE. The first follow-up visit, which occurred after a mean of 5 years after surgery, included a clinical examination, cone beam computerized tomography, and panorama or intraoral radiography. The second follow-up included panorama or intraoral radiography, and it was conducted after a mean of 8 years. RESULTS: Overall, 218 implants were placed in 127 sinuses. Nine of the 218 implants failed resulting in an overall implant survival of 95.9%. The average bone gain at the follow-up was 4.0 ±2.0 mm. CONCLUSION: The implant-supported rehabilitation achieved using graft-less LSFE was stable over time, and there was no or little impact on sinus health. Furthermore, it was concluded that the new bone formation and the amount of bone gain is proportional to the length of the implant protruding into the sinus cavity.

The effect of repeated administration of cyclophosphamide on cytochrome P450 2B in rats.

PURPOSE: The prodrug cyclophosphamide (CPA) is activated by cytochrome P450 (CYP) enzymes. CPA is one of the corner stones in all cancer treatment. We have studied the effect of repeated doses of CPA given at different time intervals on the mRNA, protein levels, and enzyme activity of CYPs in rats. EXPERIMENTAL DESIGN: Two groups of animals (A-75 and A-150) were treated with four doses of CPA (75 and 150 mg/kg, respectively) at short time intervals (6 h). The third group of animals (B-150) was treated with 150 mg/kg at 24-h intervals. Three animals were killed 30 min after administration, and three animals immediately before the next dose. RESULTS: CYP2B1 and CYP2B2 mRNAs were significantly induced at 6 h after each dose in group A-75 (maximum of 2100-fold and 60-fold after the third dose, respectively), whereas the mRNA levels measured at 6 h postadministration in group A-150 were 1,490-fold and 36-fold after the second dose. In group B-150, no significant induction of mRNA levels was observed. CYP2B1 and CYP2B2 protein levels also increased with increased mRNAs. Plasma levels of 4-hydroxy-CPA measured at 30 min after dose correlated well with the increase in protein levels. CONCLUSION: Up-regulation of CYP2B mRNA, with a concomitant increase in protein expression and activity, were observed after repeated administration of low doses of CPA compared with that found using higher doses, possibly due to toxicity counteracting induction. These results may help in designing more effective dosing schedules for CPA.

Genetic polymorphism of cytochrome P450s and P-glycoprotein in the Finnish population.

The objective of this study was to investigate the genetic polymorphism of selected cytochrome P450 (CYP) enzymes and ABCB1 (encoding P-glycoprotein) of central importance with regard to the disposition of clinically used drugs in the Finnish population and to compare the results to pre-existing data from Caucasian populations. A random sample of 449 Finns was studied. Single nucleotide polymorphisms (SNPs) were genotyped using blood-derived genomic DNA and 5'-nuclease assays. We found that the allele frequencies of CYP1A2 SNP g.-163C>A, CYP2C8*3, CYP2C9*2, CYP2C9*3 and CYP2C19*2 were similar to those seen in other Caucasian populations. However, the allelic frequency of the variant ABCB1 SNP c.3435C>T allele was lower than previously reported. The frequency of the homozygous CYP3A5*1 expression was significantly higher than expected based on Hardy-Weinberg calculations (observed n = 8 vs. expected n = 3, P = 0.01). Other genotype frequencies corresponded to the expected values. The strong linkage between the CYP2C8*3 and the CYP2C9*2 alleles was confirmed in this study and the number of individuals with the rare haplotype CYP2C8*3*3/CYP2C9*2*2 was higher than expected. We conclude that the frequency of mutated CYP alleles in Finns were in agreement with earlier findings in Caucasian populations, but a lower frequency of the ABCB1 variant allele 3435T corresponding to that reported in Asian populations was found. The higher than expected frequency of the CYP3A5*1*1 genotype and the CYP2C8*3*3/CYP2C9*2*2 haplotype may influence the response to treatment with drugs metabolized by these enzymes.

Effect of voriconazole on the pharmacokinetics of diclofenac.

The nonsteroidal anti-inflammatory drug diclofenac is extensively metabolized by cytochrome P450 (CYP) enzymes, mainly by CYP2C9. Our objective was to study the effect of voriconazole, a potent inhibitor of several CYP enzymes, on the pharmacokinetics of diclofenac. This study had a two-way, open, crossover design and included 10 healthy Caucasian male subjects. In the control phase, the subjects ingested a single 50-mg oral dose of diclofenac. In the voriconazole phase, the subjects ingested voriconazole 400 mg twice daily on the first day and 200 mg twice daily on the second day, and 50 mg diclofenac was given 1 h after the last dose of voriconazole. Plasma diclofenac concentrations were determined for up to 24 h post-dose. In the voriconazole phase, the area under the plasma concentration-time curve of diclofenac was 178% (95% CI 143-212%; P < 0.001) and the peak plasma concentration was 214% (95% CI 128-300%; P < 0.05) of the respective control value. Voriconazole did not affect significantly the elimination half-life or time to maximum concentration of diclofenac. The renal clearance of diclofenac was decreased by 47% (95% CI -76% to -16%; P < 0.01) by voriconazole. In conclusion, voriconazole increased exposure to diclofenac, probably mainly by inhibition of its cytochrome P450 (CYP)-mediated metabolism. The inhibition of CYP2C9, and to some extent that of CYP3A4 and CYP2C19 enzymes during the first-pass metabolism of diclofenac seems to be involved in the interaction. The clinical importance of the interaction between voriconazole and diclofenac remains to be studied, but lower doses of diclofenac may be adequate for patients receiving voriconazole.

Maxillary sinus augmentation using sinus membrane elevation and peripheral venous blood for implant-supported rehabilitation of the atrophic posterior maxilla: case series.

BACKGROUND: Dental implants need appropriate bone volume for adequate stability in the rehabilitation after tooth loss. In the severely atrophic posterior maxilla, the clinical success of implant treatment sometimes requires a vertical ridge augmentation in the maxillary sinus floor. PURPOSE: The purpose of this investigation was to evaluate a maxillary sinus floor augmentation technique using a replaceable bone window, elevation of the membrane, placement of implants, and injection of the patient's own venous blood to fill the voids. MATERIALS AND METHODS: Six patients with need of maxillary sinus floor augmentation participated in the study. After preparation of a replaceable bone window in the lateral aspect of the sinus and careful elevation of the Schneiderian membrane, a total of 14 Brånemark implants (TiUnite, MK III, Nobel Biocare AB, Göteborg, Sweden) were installed in the residual bone penetrating into the sinus cavity. The sinus cavity was then filled with peripheral venous blood and the bone window replaced and stabilized with a medical tissue glue (Aron Alpha A, Sankyo, Inc., Tokyo, Japan) to prevent blood leakage from the created compartment in the maxillary sinus. RESULTS: After a healing period of a minimum of 6 months, new bone was successfully generated in all 14 implant sites as judged from radiographs. One of the 14 implants failed, corresponding to a survival rate of 92.9% after a follow-up period ranging 12 to 34 months. CONCLUSIONS: The present case series demonstrate that the creation of a secluded space in the maxillary sinus and filling with venous blood results in bone formation at simultaneously installed dental implants over a 6-month period.

Reconstruction of the atrophic edentulous maxilla with free iliac crest grafts and implants: a 3-year report of a prospective clinical study.

PURPOSE: The purpose of this study was to perform a longitudinal follow-up study of implant stability in grafted maxillae with the aid of clinical, radiological, and resonance frequency analysis (RFA) parameters. MATERIALS AND METHODS: The atrophic edentulous maxillae in 29 patients were reconstructed with free iliac crest grafts using onlay/inlay or interpositional grafting techniques. The endpoint of the resorption pattern in the maxilla determined the grafting technique used. Endosteal implants were placed after 6 months of bone-graft healing. Implant stability was measured four times using RFA: when the implants were placed, after 6 to 8 months of healing, after 6 months and 3 years of bridge loading. Individual checkups were performed at the two later RFA registrations after removal of the supraconstructions (Procera Implant Bridge, Nobel Biocare AB, Göteborg, Sweden). Radiological follow up of marginal bone level was performed annually. RESULTS: Twenty-five patients remained for the follow-up period. A total of 192 implants were placed and with a survival rate of 90% at the 3-year follow up. Women and an implant position with a class 6 resorption prior to reconstruction were factors with significant increased risk for implant failure (multivariate logistic regression). Twelve of the 20 failed implants were lost before loading (early failures). The change in the marginal bone level was 0.3 +/- 0.3 mm between baseline (bridge delivery) and the 3-year follow up. The implant stability quotient (ISQ) value for all implants differed significantly between abutment connection (60.2 +/- 7.3) and after 6 months of bridge loading (62.5 +/- 5.5) (Wilcoxon signed ranks test for paired data, p=.05) but were nonsignificant between 6 months of bridge loading and 3 years of bridge loading (61.8 +/- 5.5). There was a significant difference between successful and failed implants when the ISQ values were compared for individual implants at placement (Mann-Whitney U test, p=.004). All 25 patients were provided with fixed implant bridges at the time of the 3-year follow up. CONCLUSION: This clinical follow up using radiological examinations and RFA measurements indicates a predictable and stable long-term result for patients with atrophic edentulous maxillae reconstructed with autogenous bone and with delayed placement of endosteal implants. The ISQ value at the time of placement can probably serve as an indicator of level of risk for implant failure.

Expression of cytochromes P450 3A and P-glycoprotein in human large intestine in paired tumour and normal samples.

Our objective was to investigate the expression of different cytochromes P450 3A (CYP3A4, CYP3A5, and CYP3A7) and P-glycoprotein (ABCB1) genes along the human large intestine in paired tumour and normal samples. Real-time reverse transcriptase-polymerase chain reaction was used to measure CYP3A4-, CYP3A5-, CYP3A7- and ABCB1-specific mRNA expression, and Western blot analysis was used to measure membrane protein levels of CYP3A4/7, CYP3A5 and P-glycoprotein. Levels of mRNA and membrane protein fractions in the large intestine were compared with those of normal human liver. The mRNA expressions of CYP3A4, CYP3A5, CYP3A7 and ABCB1 in the large intestine were found to be highly variable, but overall the levels were significantly lower than those measured in liver (P < 0.0001, P < 0.001, P < 0.0001 and P < 0.01, respectively). At the membrane protein level, CYP3A4/7 was detected in all large intestine samples examined and the levels were substantially higher than those of the liver (P < 0.01). Although expression of CYP3A5 was detected in all large intestine samples, in most the levels were too low to allow quantification. P-glycoprotein was readily detected at levels slightly higher than those of liver (P < 0.05). Comparison between paired samples of normal and tumour in large intestine showed no significant differences in either the mRNA or membrane protein levels of these genes. In conclusion, this work suggests a potential role of the large intestine in the absorption and metabolism of xenobiotics and nutrients and no difference in the CYP3A and P-glycoprotein membrane protein fractions and mRNA expression between normal and tumour tissues.

Bone reformation and implant integration following maxillary sinus membrane elevation: an experimental study in primates.

BACKGROUND: Recent clinical studies have described maxillary sinus floor augmentation by simply elevating the maxillary sinus membrane without the use of adjunctive grafting materials. PURPOSE: This experimental study aimed at comparing the histologic outcomes of sinus membrane elevation and simultaneous placement of implants with and without adjunctive autogenous bone grafts. The purpose was also to investigate the role played by the implant surface in osseointegration under such circumstances. MATERIALS AND METHODS: Four tufted capuchin primates had all upper premolars and the first molar extracted bilaterally. Four months later, the animals underwent maxillary sinus membrane elevation surgery using a replaceable bone window technique. The schneiderian membrane was kept elevated by insertion of two implants (turned and oxidized, Brånemark System, Nobel Biocare AB, Göteborg, Sweden) in both sinuses. The right sinus was left with no additional treatment, whereas the left sinus was filled with autogenous bone graft. Implant stability was assessed through resonance frequency analysis (Osstell, Integration Diagnostics AB, Göteborg, Sweden) at installation and at sacrifice. The pattern of bone formation in the experimental sites and related to the different implant surfaces was investigated using fluorochromes. The animals were sacrificed 6 months after the maxillary sinus floor augmentation procedure for histology and histomorphometry (bone-implant contact, bone area in threads, and bone area in rectangle). RESULTS: The results showed no differences between membrane-elevated and grafted sites regarding implant stability, bone-implant contacts, and bone area within and outside implant threads. The oxidized implants exhibited improved integration compared with turned ones as higher values of bone-implant contact and bone area within threads were observed. CONCLUSIONS: The amount of augmented bone tissue in the maxillary sinus after sinus membrane elevation with or without adjunctive autogenous bone grafts does not differ after 6 months of healing. New bone is frequently deposited in contact with the schneiderian membrane in coagulum-alone sites, indicating the osteoinductive potential of the membrane. Oxidized implants show a stronger bone tissue response than turned implants in sinus floor augmentation procedures.

Effect of caffeine intake 12 or 24 hours prior to melatonin intake and CYP1A2*1F polymorphism on CYP1A2 phenotyping by melatonin.

Earlier evidence suggests that melatonin is almost exclusively metabolised by CYP1A2 and could serve as a probe drug for CYP1A2 phenotyping. However, caffeine inhibits the metabolism of melatonin by CYP1A2 and dietary caffeine could be a potential confounder for the measurement of CYP1A2 activity with melatonin. We undertook a 3-phase cross-over study in 12 healthy volunteers to examine whether caffeine (200 mg single dose), taken 12 hr or 24 hr prior to melatonin intake, would affect the results of CYP1A2 phenotyping results as assessed by a spot sample melatonin concentration 1.5 hr after intake of 6 mg of melatonin orally. In addition we examined the influence of the CYP1A2*1F polymorphism on the phenotyping results by combining the present material with another 12 persons from a previous study. Caffeine, co-administered 12 or 24 hr prior to melatonin intake, did not have any significant effect on the 1.5 hr melatonin concentration (P=0.086 for ANOVA), but in two volunteers about 4 times increase in melatonin concentration was observed after caffeine intake 12 hr (but not 24 hr) before phenotyping with melatonin. Also, individuals homozygous for the CYP1A2*1A allele had clearly higher 1.5 hr melatonin concentration compared with the *1F/*1F or the *1F/*1A genotypes. Abstinence from caffeine for 24 hr prior to melatonin intake should be enough to overcome the possible confounding effect of caffeine on the CYP1A2 phenotyping with melatonin. Also, melatonin may be a sensitive probe to detect phenotypic differences with regard to CYP1A2*1F polymorphism. Melatonin might be, thus, advantageous for CYP1A2 phenotyping compared to the standard probe caffeine.

A histomorphometric comparison of the bone graft-titanium interface between interpositional and onlay/inlay bone grafting techniques.

PURPOSE: To analyze the bone graft-implant interface of titanium microimplants (MIs) placed at the time of bone grafting or after a healing period of 6 months and retrieved after another 6 to 14 months of healing. Integration of MIs placed in interpositional bone grafts (IBGs) in conjunction with a Le Fort I osteotomy was compared with the integration of those placed in onlay/inlay bone grafts (OBGs). MATERIALS AND METHODS: The severely atrophied edentulous maxillae of 23 patients (14 women, 9 men) were restored with autogenous bone grafts (either IBG [n=8] or OBG [n=15]) and titanium implants. Six-month periods were allowed between grafting, implant placement, and abutment connection. The bone-implant interface was studied histologically with the use of unloaded titanium MIs. RESULTS: Sixty-eight MIs were either (1) placed simultaneously with grafting and retrieved after 6, 12, or 14 months or (2) placed after 6 months of healing and retrieved after another 6 to 8 months. Histomorphometry indicated equal degrees of osseointegration for the 2 intraoral reconstruction techniques when looking at bone-implant contact, bone area in threads, and newly formed bone (NFB) (Student t test for unpaired observations). There was a significant difference between simultaneous and delayed implant placement with respect to BIC and NFB (Student t test for paired observations). Three additional MIs placed in the nongrafted residual alveolar ridge and retrieved after 6 months showed significantly more bone in threads and NFB (Student t test for paired observations; P = .003 and P = .009, respectively) compared to MIs placed at graft placement (6 months' healing). DISCUSSION: Timing of implant placement appeared more important than healing time or surgical technique. The delayed approach resulted in better implant integration, probably because of the initial revascularization of the graft. CONCLUSIONS: Implant integration was similar in the IBG and OBG groups. Placement of MIs after an initial healing period of 6 months resulted in better integration than placement simultaneously with grafting.

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape.

Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36-to-methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.