Modification of the van der Waals and Platteeuw model for gas hydrates considering multiple cage occupancy.

This study reviews available van der Waals- and Platteeuw-based hydrate models considering multiple occupancy of cavities. Small guest molecules, such as hydrogen and nitrogen, are known to occupy lattice cavities multiple times. This phenomenon has a significant impact on hydrate stability and thermodynamic properties of the hydrate phase. The objective of this work is to provide a comprehensive overview and required correlations for the implementation of a computationally sufficient cluster model that considers up to five guest molecules per cavity. Two methodologies for cluster size estimation are evaluated by existing nitrogen hydrate models showing accurate results for phase equilibria calculations. Furthermore, a preliminary hydrogen hydrate model is introduced and compared with the results of other theoretical studies, indicating that double occupancy of small sII cavities is improbable and four-molecule clusters are predominant in large sII cavities for pressures above 300 MPa. This work lays the foundation for further exploration and optimization of hydrate-based technologies for small guest molecules, e.g., storage and transportation, emphasizing their role in the future landscape of sustainable energy solutions.

Longitudinal analysis of microcirculatory parameters in gingival tissues after tooth extraction in patients with different risk profiles for wound healing disorders - a pilot study.

OBJECTIVES: We aimed to establish a risk profile for intraoral wound healing disorders based on measurements of microcirculation in gingival tissues. MATERIALS AND METHODS: Oxygen saturation (SO2) and blood flow in gingival tissues were measured with tissue spectrometry and laser doppler spectroscopy in 37 patients before/after tooth extractions. Patients were assigned to four groups: anamnestically and periodontally healthy patients (n = 7), anamnestically healthy but suffering from periodontitis (n = 10), anamnestically healthy but smoking and suffering from periodontitis (n = 10) and suffering from diabetes and periodontitis (n = 10). Measurements were performed at three different time points: Baseline measurement (T0), one day post extractionem (p.e.) (T1) and seven days p.e. (T2). RESULTS: Baseline SO2 values were higher in control patients (p = .038). This effect was most evident in comparison to smokers suffering from periodontitis (p = .042), followed by diabetics suffering from periodontitis (p = .09). An opposite trend was seen for blood flow. Patients suffering from periodontitis demonstrated higher blood flow values (p = .012). Five patients, which belonged to the group of smokers suffering from periodontitis, showed clinically a delayed wound healing. CONCLUSION: Differences in SO2 and blood flow of gingival tissue could be detected in different groups of patients with existing periodontitis compared to control patients. CLINICAL RELEVANCE: Lower baseline SO2 values could be a warning signal for possible wound healing disorders after oral surgery.

Clinical implementation of axial angulation of incisors in the course of routine fixed appliance treatment - a retrospective cohort study.

PURPOSE: To identify clinically relevant factors for changes in axial angulation of incisors during routine fixed appliance orthodontic treatment. METHODS: A total of 106 patients (grades 1-2 of IOTN, 64 females, 42 males; mean age: 15.5 years) from a private practice and treated with metal or ceramic brackets were included in this retrospective cohort study. The axial angulation of the upper and lower incisors was measured on lateral cephalograms before insertion of the first rectangular 0.016 × 0.022-in NiTi archwire (T0) and at the end of treatment about 8 weeks after insertion of the working 0.019 × 0.025-in stainless steel archwire (T1). Treatment-related changes according to bracket type, initial situation, premolar extraction, angle class, and skeletal vertical configuration were analyzed. RESULTS: Although statistically significant treatment-related changes were seen for both the upper incisors (+ 1.3°) and the lower incisors (- 5.2°), only in ten patients (9.4%) was the prescribed torque value of 17° for the upper incisors and in no patient for the lower incisors achieved. A negative association between the induced change of axial angulation of incisors and the initial values was detected for the upper incisors as well as for the lower incisors. A comparison of the angle classes revealed significant differences in incisor changes. At the end of therapy, only a slight change for the upper central incisors in patients in angle class I cases and a significantly greater change in patients with angle class II/2 was observed. Cases with premolar extraction ended with lower axial angulation of the incisor than cases without extraction. The individual analysis of possible influencing factors also revealed an association with the vertical skeletal configuration. CONCLUSIONS: For the first time, the presented data show clinically relevant influencing factors for incisor axial angulation changes of the upper and lower incisors in relation to the torque value of the applied brackets in the course of routine clinical practice. For the orthodontist, it remains mandatory to decide whether a customized system must be individualized in order to achieve individual therapy goals.

The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides.

In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions.

Post-treatment Stability in Orthodontic Retention with Twistflex Retainers-Do Patients Benefit from Additional Removable Retainers?

OBJECTIVES: To evaluate post-treatment movements of lower anterior teeth during orthodontic retention in patients with fixed twistflex retainers versus those with combined fixed and removable retainers. MATERIALS AND METHODS: This study was based on a retrospective data analysis of 57 adult patients during orthodontic retention. They were assigned to two groups: In group 1 (n = 30) the lower jaw was provided with twistflex retainers only and in group 2 (n = 27) with a twistflex combined with a removable retainer for night-time use. Orthodontic study models of the lower jaw were digitalized and superimposed. Tooth movements were analyzed at the retainer bonding (t0) and follow-up appointment ≥ six months later (t1). Rotational tooth movements (°) were measured around the x-axis (mesial/distal direction), the y-axis (buccal/lingual direction) and the z-axis (longitudinal direction, tooth axis). Translational tooth movements (mm) were registered along the x-axis (buccal/lingual direction), the y-axis (mesial/distal direction) and the z-axis (apical/coronal direction). RESULTS: Canine and incisor position changes during orthodontic retention were more pronounced in group 1 compared to group 2 except for canine rotations around the z-axis. In both groups in most of the cases stable lower incisor alignment could be found, but the proportion was significant higher in group 2 (group 1: 56.7% vs. group 2: 81.5%). Severe misalignment was present in 13.3% of the participants of group 1 and only in 7.4% of group 2. The extent of canine tipping and movements along the x- and y-axis in severe misalignment cases was significantly lower in group 2 compared to 1. CONCLUSIONS: Lower incisor alignment was more stable in patients with combined fixed and removable retainers compared to fixed retainers only. CLINICAL RELEVANCE: Based on the present findings, the routinely application of supplementary removable retainers can be recommended to enhance anterior tooth alignment in patients with fixed twistflex retainers.

Interaction of periodontitis and orthodontic tooth movement-an in vitro and in vivo study.

OBJECTIVES: The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). MATERIALS AND METHODS: The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. RESULTS: Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. CONCLUSIONS: Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. CLINICAL RELEVANCE: Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides.

sRNAs have an important role in the regulation of bacterial gene expression. The sRNA, UdsC, of Rhodobacter sphaeroides is derived from the 3' UTR of the RSP_7527 mRNA, which encodes a hypothetical protein. Here, we showed the effect of UdsC on the resistance of Rhodobacter sphaeroides to hydrogen peroxide and on its motility. In vitro binding assays supported the direct interaction of UdsC with the 5' UTR of the rpoHII mRNA. RpoHII is an alternative sigma factor with an important role in stress responses in R. sphaeroides, including its response to hydrogen peroxide. We also demonstrated that RpoHII controls the expression of the torF gene, which encodes an important regulator of motility genes. This strongly suggested that the observed effect of UdsC on TorF expression is indirect and mediated by RpoHII.

Extending the allelic spectrum at noncoding risk loci of orofacial clefting.

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Interplay between formation of photosynthetic complexes and expression of genes for iron-sulfur cluster assembly in Rhodobacter sphaeroides?

Formation of photosynthetic complexes leads to a higher demand for Fe-S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium Rhodobacter sphaeroides expression of the isc-suf operon for Fe-S cluster formation may be increased under conditions that promote formation of photosynthetic complexes and that, vice versa, lack of the IscR regulator may also affect photosynthesis gene expression. To test this hypothesis, we monitored the activities of the isc-suf sense and anti-sense promoters under different growth conditions and in mutants which are impaired in formation of photosynthetic complexes. We also tested expression of photosynthesis genes in a mutant lacking the IscR regulator. Our results are not in agreement with a co-regulation of the Isc-Suf system and the photosynthetic apparatus at level of transcription. We provide evidence that, coordination of the systems occurs at post-transcriptional levels. Increased levels of isc-suf mRNAs under conditions promoting formation of photosynthetic complexes are due to higher RNA stability.

The effect of timing of orthodontic therapy on the outcomes of regenerative periodontal surgery in patients with stage IV periodontitis: A multicenter randomized trial.

AIM: To compare the outcomes after early (4 weeks post surgery) or late (6 months post surgery) orthodontic therapy (OT) following regenerative surgery of intra-bony defects (IDs). MATERIALS AND METHODS: In a multi-center, parallel-group, randomized clinical trial, 43 patients with stage IV periodontitis were randomized to receive either early (n = 23) or late OT (n = 20) following regenerative surgery of IDs. Primary outcome was change in clinical attachment level (CAL) in one target ID at 12 months after surgery. Secondary outcomes were changes of probing pocket depth (PPD), bleeding on probing (BOP), and frequency of pocket closure. RESULTS: No statistically significant differences between groups could be observed for CAL gain (5.4 mm [±2.1 mm] for early; 4.5 mm [±1.7 mm] for late OT). PPD was reduced by 4.2 mm (±1.9 mm) in the early group and by 3.9 mm (±1.5 mm) in the late group (p > .05). Pocket closure (PPD ≤ 4 mm) was obtained in 91% of defects in early compared to 85% in late OT. CONCLUSION: In the inter-disciplinary treatment of periodontitis stage IV, OT can be initiated already 4 weeks after regenerative surgery of IDs with favourable results, thus reducing the overall treatment time.

Dose rate correction for a silicon diode detector array.

PURPOSE: A signal dependence on dose rate was reported for the ArcCHECK array due to recombination processes within the diodes. The purpose of our work was to quantify the necessary correction and apply them to quality assurance measurements. METHODS: Static 10 × 10 cm2 6-MV fields delivered by a linear accelerator were applied to the detector array while decreasing the average dose rate, that is, the pulse frequency, from 500 to 30 MU/min. An ion chamber was placed inside the ArcCHECK cavity as a reference. Furthermore, the instantaneous dose rate dependence (DRD) was studied. The position of the detector was adjusted to change the dose-per-pulse, varying the distance between the focus and the diode closest to the focus between 69.6 and 359.6 cm. Reference measurements were performed with an ion chamber placed inside a PMMA slab phantom at the same source-to-detector distances ( S D D s ) . Exponential saturation functions were fitted to the data, with different parameters to account for two generations of ArcCHECK detectors (types 2 and 3) and both DRDs. Corrections were applied to 12 volumetric modulated arc therapy plans. RESULTS: The sensitivity decreased by up to 2.8% with a decrease in average dose rate and by 9% with a decrease in instantaneous dose rate. Correcting the average DRD, the mean gamma pass rates (2%/2-mm criterion) of the treatment plans were improved by 5 percentage points (PP) for diode type 3 and 0.4 PP for type 2. Correcting the instantaneous DRD, the improvement was 8.4 PP for type 3 and 0.9 PP for type 2. CONCLUSIONS: The instantaneous DRD was identified as the prevailing effect on the diode sensitivity. We developed and validated a method to correct this behavior. The number of falsely not passed treatment plans could be considerably reduced.

A Complex Network of Sigma Factors and sRNA StsR Regulates Stress Responses in R. sphaeroides.

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.

Regulation of Anti-Apoptotic SOD2 and BIRC3 in Periodontal Cells and Tissues.

The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum, but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.

Resistin Is Increased in Periodontal Cells and Tissues: In Vitro and In Vivo Studies.

Resistin, a proinflammatory adipokine, is elevated in many inflammatory diseases. However, little is known about its performance in periodontitis. The present study is aimed at evaluating resistin expression and synthesis in periodontal cells and tissues under inflammatory/microbial stress in addition to its effects on the periodontium. In vivo, 24 male rats were randomly divided into two groups: control and ligature-induced periodontal disease. After 6 and 12 days, animals were sacrificed to analyze gene expression of adipokines, bone loss, inflammation, and resistin synthesis. In vitro, human periodontal ligament (PDL) fibroblasts were used to evaluate the expression of resistin after inflammatory stimuli. In addition, PDL fibroblasts were exposed to resistin to evaluate its role on soft and hard tissue metabolism markers. The periodontitis group demonstrated significant bone loss, an increase in the number of inflammatory cells and vascular structures, an increase in resistin expression and synthesis, and a decrease in the expression of adiponectin, leptin, and its functional receptor. PDL fibroblasts showed a significant increase in resistin expression and synthesis in response to the inflammatory stimulus by IL-1ß. Resistin induced an increase in cytokine expression and a decrease in the regulation of some hard tissue and matrix formation genes in PDL fibroblasts. These data indicate that resistin is produced by periodontal cells and tissues, and this effect is enhanced by inflammatory stimuli. Moreover, resistin seems to interfere with soft and hard tissue metabolism during periodontitis by reducing markers related to matrix formation and bone tissue.

Orthodontic cell stress modifies proinflammatory cytokine expression in human PDL cells and induces immunomodulatory effects via TLR-4 signaling in vitro.

OBJECTIVE: Biomechanical orthodontics loading of the periodontium initiates a cascade of inflammatory signaling events that induce periodontal remodeling and finally facilitate orthodontic tooth movement. Pattern recognition receptors such as toll-like receptors (TLRs) have been well characterized for their ability to induce the activation of inflammatory, immunomodulatory cytokines. Here, we examined whether the cellular response of human periodontal ligament (hPDL) cells to mechanical stress involves TLR-4 signaling in vitro. MATERIALS AND METHODS: Confluent hPDL cells were cultured in the presence of 5 µg/ml TLR-4 antibody (TLR-4ab) for 1 h prior to the induction of compressive forces by the use of round glass plates for 24 h. At harvest, interleukin-6 and interleukin-8 (IL-6, IL-8) mRNA and protein expression were analyzed by real-time PCR and ELISA. The immunomodulatory role of mechanical cell stress and TLR-4 signaling was addressed in co-culture experiments of hPDL and THP-1 cells targeting monocyte adhesion and by culturing osteoclastic precursors (RAW 264.7) in the presence of the conditioned medium of hPDL cells that had been mechanically loaded before. RESULTS: Basal expression of IL-6 and IL-8 was not affected by TLR-4ab, but increased significantly upon mechanical loading of hPDL cells. When cells were mechanically stressed in the presence of TLR-4ab, the effect seen for loading alone was markedly reduced. Likewise, monocyte adhesion and osteoclastic differentiation were enhanced significantly by mechanical stress of hPDL cells and this effect was partially inhibited by TLR-4ab. CONCLUSIONS: The results of the present study indicate a proinflammatory and immunomodulatory influence of mechanical loading on hPDL cells. Intracellular signaling involves a TLR-4-dependent pathway. CLINICAL RELEVANCE: These findings hold out the prospect of interfering with the cellular response to mechanical cell stress in order to minimize undesired side effects of orthodontic tooth movement.

CXCL1, CCL2, and CCL5 modulation by microbial and biomechanical signals in periodontal cells and tissues-in vitro and in vivo studies.

OBJECTIVES: This study was established to investigate whether the chemokines CXCL1, CCL2, and CCL5 are produced in periodontal cells and tissues and, if so, whether their levels are regulated by microbial and/or mechanical signals. MATERIALS AND METHODS: The chemokine expression and protein levels in gingival biopsies from patients with and without periodontitis were analyzed by RT-PCR and immunohistochemistry. The chemokines were also analyzed in gingival biopsies from rats subjected to experimental periodontitis and/or orthodontic tooth movement. Additionally, chemokine levels were determined in periodontal fibroblasts exposed to the periodontopathogen Fusobacterium nucleatum and mechanical forces by RT-PCR and ELISA. RESULTS: Higher CXCL1, CCL2, and CCL5 levels were found in human and rat gingiva from sites of periodontitis as compared with periodontally healthy sites. In the rat experimental periodontitis model, the bacteria-induced upregulation of these chemokines was significantly counteracted by orthodontic forces. In vitro, F. nucleatum caused a significant upregulation of all chemokines at 1 day. When the cells were subjected simultaneously to F. nucleatum and mechanical forces, the upregulation of chemokines was significantly inhibited. The transcriptional findings were paralleled at protein level. CONCLUSIONS: This study provides original evidence in vitro and in vivo that the chemokines CXCL1, CCL2, and CCL5 are regulated by both microbial and mechanical signals in periodontal cells and tissues. Furthermore, our study revealed that biomechanical forces can counteract the stimulatory actions of F. nucleatum on these chemokines. CLINICAL RELEVANCE: Mechanical loading might aggravate periodontal infection by compromising the recruitment of immunoinflammatory cells.

Regulation of Autophagic Signaling by Mechanical Loading and Inflammation in Human PDL Fibroblasts.

Autophagy (cellular self-consumption) is a crucial adaptation mechanism during cellular stress conditions. This study aimed to examine how this important process is regulated in human periodontal ligament (PDL) fibroblasts by mechanical and inflammatory stress conditions and whether the mammalian target of rapamycin (mTOR) signaling pathway is involved. Autophagy was quantified by flow cytometry. Qualitative protein phosphorylation profiling of the mTOR pathway was carried out. Effects of mTOR regulation were assessed by quantification of important synthesis product collagen 1, cell proliferation and cell death with real-time PCR and flow cytometry. Autophagy as a response to mechanical or inflammatory treatment in PDL fibroblasts was dose and time dependent. In general, autophagy was induced by stress stimulation. Phosphorylation analysis of mTOR showed regulatory influences of mechanical and inflammatory stimulation on crucial target proteins. Regulation of mTOR was also detectable via changes in protein synthesis and cell proliferation. Physiological pressure had cell-protective effects (p = 0.025), whereas overload increased cell death (p = 0.003), which was also promoted in long-term inflammatory treatment (p < 0.001). Our data provide novel insights about autophagy regulation by mechanical and inflammatory stress conditions in human PDL fibroblasts. Our results suggest some involvement of the mTOR pathway in autophagy and cell fate regulation under the named conditions.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Autophagy in periodontal ligament fibroblasts under biomechanical loading.

Autophagy (cellular self-consumption) is an adaptive stress response and an important aspect of adaption to mechanical loading. If mechanical forces are associated with autophagy regulation in periodontal ligament (PDL) fibroblasts is still unknown. The aim of this study was to analyze the influence of force magnitude on autophagy regulation and subsequently on cell death in human PDL fibroblasts. Autophagy-associated genes were analyzed with a specific PrimePCR assay after 24 h of stimulation with high (STSH) and low magnitudes (STSL) of static tensile strain applied to PDL fibroblasts. Based on the results, targets were selected for further real-time PCR analysis. The autophagic flux was assessed by immunoblotting for autophagy marker microtubule-associated protein 1, light chain 3, and by autophagosome staining. Cell death was determined by TUNEL assay and Cell Death Detection ELISAPLUS. Autophagy was induced pharmacologically by rapamycin and inhibited by chloroquine. For statistical analysis, the Kruskal Wallis test followed by the post-hoc Dunnett's test was used. Static tensile strain had regulatory effects on mRNA expression of multiple autophagy-associated targets. Stimulation with STSH induced mRNA expression changes in more autophagy-associated targets than STSL. The autophagic flux was induced by STSH while STSL had no significant effect on autophagosome formation. Furthermore, autophagy inhibition led to increased cell death. Low magnitudes of tensile strain seem to have cell-protective properties. Taken together, our findings provide novel insights about autophagy regulation by biomechanical loading in human PDL fibroblasts. Our results suggest a gradual response of autophagy to static tensile strain in human PDL fibroblasts.

Regulation of tyrosine hydroxylase in periodontal fibroblasts and tissues by obesity-associated stimuli.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamines and has been connected to aggravated progression of periodontal disease under chronic stress. Obesity is known to increase the risk of periodontitis and adipokines have been suggested to be a pathomechanistic link. This study examines if obesity-associated stimuli have regulatory effects on TH levels in periodontal cells and tissues. Human periodontal ligament fibroblasts were cultured in the presence of leptin or visfatin for up to 2 days. Untreated cells served as control. TH regulation was analyzed by real-time PCR, immunocytochemistry and ELISA. TH gene expression in periodontal tissues of normal-weight and obese rodents was determined. Examination of gingival biopsies from rats and patients with and without periodontal disease was performed by real-time PCR or immunohistochemistry. For statistics, ANOVA and post hoc tests were applied (p < 0.05). In vitro, TH gene expression and protein levels were increased by leptin and visfatin. In vivo, TH gene expression was upregulated in periodontal tissues of obese rodents as compared to normal-weight animals. Additionally, increased TH gene expression was found in rat gingival biopsies with experimental periodontitis. Human gingival biopsies from sites of periodontitis confirmed the animal data by demonstrating elevated TH levels at periodontally diseased sites. This study provides original evidence that obesity-associated stimuli induce a TH upregulation in periodontal cells and tissues. Since TH levels were also increased at periodontitis sites, our in vitro and animal findings suggest that this enzyme could represent a pathomechanism whereby obesity contributes to periodontitis.