Network-based prioritization and validation of regulators of vascular smooth muscle cell proliferation in disease.

Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation characterize vascular diseases causing heart attack and stroke. Here we elucidate molecular determinants governing VSMC proliferation by reconstructing gene regulatory networks from single-cell transcriptomics and epigenetic profiling. We detect widespread activation of enhancers at disease-relevant loci in proliferation-predisposed VSMCs. We compared gene regulatory network rewiring between injury-responsive and nonresponsive VSMCs, which suggested shared transcription factors but differing target loci between VSMC states. Through in silico perturbation analysis, we identified and prioritized previously unrecognized regulators of proliferation, including RUNX1 and TIMP1. Moreover, we showed that the pioneer transcription factor RUNX1 increased VSMC responsiveness and that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signaling. Both RUNX1 and the TIMP1-CD74 axis were expressed in human VSMCs, showing low levels in normal arteries and increased expression in disease, suggesting clinical relevance and potential as vascular disease targets.

Genetic influence on vascular smooth muscle cell apoptosis.

Vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis play important roles in many physiological processes and pathological conditions. To identify genetic influences on VSMC behavior, we measured these traits and undertook genome-wide association studies in primary umbilical artery-derived VSMCs from >2000 individuals. Although there were no genome-wide significant associations for VSMC proliferation or migration, genetic variants at two genomic loci (7p15.3 and 7q32.3) showed highly significant associations with VSMC apoptosis (P = 1.95 × 10-13 and P = 7.47 × 10-9, respectively). The lead variant at the 7p51.3 locus was associated with increased expression of the GSDME and PALS2 genes in VSMCs. Knockdown of GSDME or PALS2 in VSMCs attenuated apoptotic cell death. A protein co-immunoprecipitation assay indicated that GSDME complexed with PALS2. PALS2 knockdown attenuated activated caspase-3 and GSDME fragmentation, whilst GSDME knockdown also reduced activated caspase-3. These findings provide new insights into the genetic regulation of VSMC apoptosis, with potential utility for therapeutic development.

Effects of Coronary Artery Disease-Associated Variants on Vascular Smooth Muscle Cells.

BACKGROUND: Genome-wide association studies have identified many genetic loci that are robustly associated with coronary artery disease (CAD). However, the underlying biological mechanisms are still unknown for most of these loci, hindering the progress to medical translation. Evidence suggests that the genetic influence on CAD susceptibility may act partly through vascular smooth muscle cells (VSMCs). METHODS: We undertook genotyping, RNA sequencing, and cell behavior assays on a large bank of VSMCs (n>1499). Expression quantitative trait locus and splicing quantitative trait locus analyses were performed to identify genes with an expression that was influenced by CAD-associated variants. To identify candidate causal genes for CAD, we ascertained colocalizations of VSMC expression quantitative trait locus signals with CAD association signals by performing causal variants identification in associated regions analysis and the summary data-based mendelian randomization test. Druggability analysis was then performed on the candidate causal genes. CAD risk variants were tested for associations with VSMC proliferation, migration, and apoptosis. Collective effects of multiple CAD-associated variants on VSMC behavior were estimated by polygenic scores. RESULTS: Approximately 60% of the known CAD-associated variants showed statistically significant expression quantitative trait locus or splicing quantitative trait locus effects in VSMCs. Colocalization analyses identified 84 genes with expression quantitative trait locus signals that significantly colocalized with CAD association signals, identifying them as candidate causal genes. Druggability analysis indicated that 38 of the candidate causal genes were druggable, and 13 had evidence of drug-gene interactions. Of the CAD-associated variants tested, 139 showed suggestive associations with VSMC proliferation, migration, or apoptosis. A polygenic score model explained up to 5.94% of variation in several VSMC behavior parameters, consistent with polygenic influences on VSMC behavior. CONCLUSIONS: This comprehensive analysis shows that a large percentage of CAD loci can modulate gene expression in VSMCs and influence VSMC behavior. Several candidate causal genes identified are likely to be druggable and thus represent potential therapeutic targets.

Programmed Cell Death in Developing Brachypodium distachyon Grain.

The normal developmental sequence in a grass grain entails the death of several maternal and filial tissues in a genetically regulated process termed programmed cell death (PCD). The progression and molecular aspects of PCD in developing grains have been reported for domesticated species such as barley, rice, maize and wheat. Here, we report a detailed investigation of PCD in the developing grain of the wild model species Brachypodium distachyon. We detected PCD in developing Brachypodium grains using molecular and histological approaches. We also identified in Brachypodium the orthologs of protease genes known to contribute to grain PCD and surveyed their expression. We found that, similar to cereals, PCD in the Brachypodium nucellus occurs in a centrifugal pattern following anthesis. However, compared to cereals, the rate of post-mortem clearance in the Brachypodium nucellus is slower. However, compared to wheat and barley, mesocarp PCD in Brachypodium proceeds more rapidly in lateral cells. Remarkably, Brachypodium mesocarp PCD is not coordinated with endosperm development. Phylogenetic analysis suggests that barley and wheat possess more vacuolar processing enzymes that drive nucellar PCD compared to Brachypodium and rice. Our expression analysis highlighted putative grain-specific PCD proteases in Brachypodium. Combined with existing knowledge on grain PCD, our study suggests that the rate of nucellar PCD moderates grain size and that the pattern of mesocarp PCD influences grain shape.

Regenerative Endodontic Therapy in Mature Teeth Using Human-Derived Composite Amnion-Chorion Membrane as a Bioactive Scaffold: A Pilot Animal Investigation.

INTRODUCTION: Human-derived composite amnion-chorion membrane (ACM) has been used for various regenerative treatments. The aim of this pilot study was to investigate the effectiveness of the ACM as a scaffold for pulp regeneration in mature canine teeth. METHODS: A total of 24 roots from mature premolars in dogs were included for regenerative procedures using blood clots (BC) (group 1, n = 8), collagen membrane (CM) (group 2, n = 8), and ACM (group 3, n = 8). Each tooth was left open through a buccal access to induce root canal infection and inflammation. The root canals were disinfected with 1.5% NaOCl and calcium hydroxide intracanal medicament. After 2 weeks, bleeding was evoked to induce blood clot formation (group 1) or before the placement of the membranes (groups 2 and 3). After 12 weeks, the animals were euthanized for histologic assessment. The histologic data including intracanal fibrous connective tissue, odontoblast-like cell lining, intracanal mineralized tissue, periapical inflammation, and apical closure were qualitatively and quantitively analyzed. RESULTS: Histologic analysis revealed that intracanal fibrous connective tissue was identified in all groups, but a higher volume of the fibrous tissues was formed in the ACM group. Odontoblast-like cells were only observed in the ACM group. The intracanal mineralized tissue was observed only in the BC and CM groups. The BC group showed more periapical inflammation than the ACM group (P < .05). Apical closure was more often found in the CM group than the BC group (P < .05). CONCLUSIONS: More intracanal fibrous tissue and odontoblast-like cell lining, and less periapical inflammation were observed after regenerative endodontic treatment in mature teeth using the ACM than blood clot alone or blood clot with collagen membrane. The use of the ACM may be useful for a cell-homing-based pulp regeneration in mature teeth.

APETALA2 functions as a temporal factor together with BLADE-ON-PETIOLE2 and MADS29 to control flower and grain development in barley.

Cereal grain develops from fertilised florets. Alterations in floret and grain development greatly influence grain yield and quality. Despite this, little is known about the underlying genetic control of these processes, especially in key temperate cereals such as barley and wheat. Using a combination of near-isogenic mutant comparisons, gene editing and genetic analyses, we reveal that HvAPETALA2 (HvAP2) controls floret organ identity, floret boundaries, and maternal tissue differentiation and elimination during grain development. These new roles of HvAP2 correlate with changes in grain size and HvAP2-dependent expression of specific HvMADS-box genes, including the B-sister gene, HvMADS29 Consistent with this, gene editing demonstrates that HvMADS29 shares roles with HvAP2 in maternal tissue differentiation. We also discovered that a gain-of-function HvAP2 allele masks changes in floret organ identity and grain size due to loss of barley LAXATUM.A/BLADE-ON-PETIOLE2 (HvBOP2) gene function. Taken together, we reveal novel pleiotropic roles and regulatory interactions for an AP2-like gene controlling floret and grain development in a temperate cereal.

LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat.

Mutations at the LYS3 locus in barley have multiple effects on grain development, including an increase in embryo size and a decrease in endosperm starch content. The gene underlying LYS3 was identified by genetic mapping and mutations in this gene were identified in all four barley lys3 alleles. LYS3 encodes a transcription factor called Prolamin Binding Factor (PBF). Its role in controlling embryo size was confirmed using wheat TILLING mutants. To understand how PBF controls embryo development, we studied its spatial and temporal patterns of expression in developing grains. The PBF gene is expressed in both the endosperm and the embryos, but the timing of expression in these organs differs. PBF expression in wild-type embryos precedes the onset of embryo enlargement in lys3 mutants, suggesting that PBF suppresses embryo growth. We predicted the down-stream target genes of PBF in wheat and found them to be involved in a wide range of biological processes, including organ development and starch metabolism. Our work suggests that PBF may influence embryo size and endosperm starch synthesis via separate gene control networks.

A Novel Approach to Fracture Resistance Using Horizontal Posts after Endodontic Therapy: A Case Report and Review of Literature.

Studies of endodontic outcomes suggest that fracture of the tooth after endodontic therapy may be a greater problem than endodontic reinfection. Immediate full coverage with or without a post and core is the best way to prevent fracture. Unfortunately, in many population areas, due predominantly to cost, this restoration is often delayed, leading to fracture of the tooth. Reviews of several in vitro studies suggest that a composite restoration reinforced with glass fibers, particularly with fiberglass posts laid horizontally in a buccolingual direction, significantly increased the resistance to fracture. This case report describes in detail the placement of horizontal posts in an endodontically treated molar to reinforce the coronal structure. In the discussion, we outlined the agreement of most of the authors on the increase in fracture resistance and the lack of consensus on what constitutes a catastrophic fracture.

Besides and Beyond Flowering: Other roles of EuAP2 Genes in Plant Development.

EuAP2 genes are well-known for their role in flower development, a legacy of the founding member of this subfamily of transcription factors, whose mutants lacked petals in Arabidopsis. However, studies of euAP2 genes in several species have accumulated evidence highlighting the diverse roles of euAP2 genes in other aspects of plant development. Here, we emphasize other developmental roles of euAP2 genes in various species and suggest a shift from regarding euAP2 genes as just flowering genes to consider the global role they may be playing in plant development. We hypothesize that their almost universal expression profile and pleiotropic effects of their mutation suggest their involvement in fundamental plant development processes.

Retrospective Evaluation of Treatment Planning for Dental Implant.

This retrospective study investigates the diagnostic rationale for the extraction of teeth and their replacement with implants in a dental school setting. Most of the teeth were extracted for restorative reasons (62.7%). The other reasons for extraction were periodontal (35.1%) and endodontic (1.3%). A panel of endodontists disagreed with the treatment-planning dentists' decisions in 40.3% of the cases. Slightly more than half, 52.9%, of the disagreements were for restorative reasons. Most of the decisions in disagreement were made by general dentists (60.6%), far fewer by prosthodontists (25.5%), periodontists (12.2%), and oral surgeons (1.6%). An extensive review of the literature is provided.

Prevalence of middle mesial canals in mandibular molars after guided troughing under high magnification: an in vivo investigation.

INTRODUCTION: A limited number of in vivo studies have discussed the prevalence of middle mesial canals in root canal systems of mandibular molars. The reported results have varied between 1% and 25%, with no detailed description of the depth and direction of troughing needed to identify such small canal orifices. The objective of the present study was to determine (1) the prevalence of a middle mesial canal before and after troughing by using a standardized troughing technique, (2) the pathway of the middle mesial canal in relation to the mesiobuccal (MB) and mesiolingual (ML) canals, and (3) its correlation with the patient's age. METHODS: Ninety-one mandibular molars from 87 patients were included in this study. The patient's age and tooth number were recorded. After access cavity preparation, a standardized troughing technique was performed between MB and ML canals to search for a middle mesial canal by using a dental operating microscope. If a middle mesial canal was located, it was recorded as separate or as joining the MB or the ML canals. Results were statistically analyzed by using Z test and logistic regression. RESULTS: A middle mesial canal was found in 42 of 91 mandibular molars (46.2%). Six middle mesial canals were located after conventional access preparation (6.6%). The other 36 were located after standardized troughing (39.6%). The results were statistically significant (P < .001). There was a higher tendency to locate the middle mesial canal in second molars (60%) versus first molars (37.5%). Younger patients had a significantly higher incidence of a middle mesial canal (P = .004). CONCLUSIONS: The middle mesial canal was present in 46.2% of mandibular molars. High magnification, troughing, and patient's age appeared to be determining factors in accessing the middle mesial canal.

Guided tissue regeneration in endodontic surgery by using a bioactive resorbable membrane.

INTRODUCTION: Guided tissue regeneration is a valuable technique available to the endodontist because the quality, quantity, or extent of bone loss cannot be visualized by the surgeon until the tissue is reflected and the surgical site is exposed. METHODS: After repeated attempts at nonsurgical treatment, a patient with a recurring sinus tract over the distobuccal root of an upper molar ultimately had the distobuccal root resected, leaving a 10 × 10 mm bony defect. This dehiscence was filled with freeze-dried bone and covered with a flexible and absorbable bioactive membrane that was new to endodontics. RESULTS: Healing was uneventful, and bone regeneration was rapid and extensive as observed at the time of a second surgery just 5 months later. This can be attributed at least in part to the use of the bioactive membrane that contains an array of growth factors that enhance cell proliferation, inflammation, recruitment of progenitor cells, and metalloproteinase activity. CONCLUSIONS: The use of the bioactive membrane in endodontic surgery should be considered to best restore the attachment apparatus to the tooth and prevent the downgrowth of a long junctional epithelium. The endodontist's attention must not be limited to the apical region alone.

Schadenfreude: an all too common affliction.

The ADA Code of Professional Conduct requires that care be taken that criticism of colleagues' work is justifiable. Several cases are presented where this standard appeared not to have been met, and the consequences were dire for all involved. Sometimes unjustified criticism can be as inadvertent as ambiguous body language; sometimes it is possible to interpret unjustified criticism as being driven by envy or by what the Germans call schadenfreude--satisfaction at others' misfortunes.

Effects of growth factors on dental stem/progenitor cells.

The primary goal of regenerative endodontics is to restore the vitality and functions of the dentin-pulp complex, as opposed to filing of the root canal with bioinert materials. A myriad of growth factors regulates multiple cellular functions including migration, proliferation, differentiation, and apoptosis of several cell types intimately involved in dentin-pulp regeneration. Recent work showing that growth factor delivery, without cell transplantation, can yield pulp-dentin-like tissues in vivo provides one of the tangible pathways for regenerative endodontics. This review synthesizes knowledge on many growth factors that are known or anticipated to be efficacious in dental pulp-dentin regeneration.

Cost-effectiveness of endodontic molar retreatment compared with fixed partial dentures and single-tooth implant alternatives.

INTRODUCTION: One of the most challenging situations in dentistry is a failed root canal treatment case. Should a failed root canal-treated tooth be retreated nonsurgically or surgically, or should the tooth be extracted and replaced with an implant-supported restoration or fixed partial denture? These four treatment alternatives were compared from the perspective of cost-effectiveness on the basis of the current best available evidence. METHODS: The costs of the four major treatment modalities were calculated using the national fee averages from the 2009 American Dental Association survey of dental fees. The outcome data of all treatment modalities were retrieved from meta-analyses after electronic and manual searches were undertaken in the database from MEDLINE, Cochrane, ISI Web of Knowledge, and Scopus up to April 2010. The treatment strategy model was built and run with TreeAge decision analysis software (TreeAge Software, Inc, Williamstown, MA). RESULTS: Endodontic microsurgery was the most cost-effective approach followed by nonsurgical retreatment and crown, then extraction and fixed partial denture, and finally extraction and single implant-supported restoration. CONCLUSIONS: The cost-effectiveness analysis showed that endodontic microsurgery was the most cost-effective among all the treatment modalities for a failed endodontically treated first molar. A single implant-supported restoration, despite its high survival rate, was shown to be the least cost-effective treatment option based on current fees.

Treatment planning considerations for the endodontically compromised tooth.

Recent advances in microendodontic procedures have dramatically enhanced our ability to successfully retreat most endodontic failures, either surgically or nonsurgically. Ironically, this comes at a time when we are seeing an increasing tendency to forego heroic endodontic procedures in favor of a single-tooth implant. While an implant has proved to be a reliable replacement for a missing tooth, it should not be considered the true equivalent of a functional natural tooth. Whenever the possibility of retaining a functional tooth exists, it should be considered seriously before an ultimate decision is made.