Efficiency and Predictability of Coronal Maxillary Expansion Repercussion with the Aligners System: A Retrospective Study.

The Invisalign® system (SmartForce® G8) aims to guarantee aesthetics and provide good orthodontic treatment results. Dentoalveolar expansion is possible with clear aligners and can be used to correct dentoalveolar crossbite, resolve crowding or modify the arch shape. Despite the treatment's effectiveness, there is still disagreement among professionals concerning its true clinical potential. This study aimed to analyze the effectiveness and predictability of coronal tooth expansion movement in permanent dentition in patients who had completed the first phase of treatment with Invisalign® orthodontic aligners. MATERIALS AND METHODS: The tooth movement tables of 75 previously selected cases were analyzed in terms of dental-arch width and expansion efficiency, through the Invisalign® platform, considering the pre-treatment (T0), planned treatment (TP) and post-treatment models (T1) using ClinCheck Pro® 6.0 software. All patients were treated by an orthodontic specialist and Invisalign® Diamond Provider in a private practice (T.P.). RESULTS: Difference between T1 and T0: for each maxillary and mandibular measurement, there was a statistically significant difference between pre- and post-aligner treatment values. The greatest amount of expansion occurred in both the upper and the lower premolars. Difference between TP and T1: for each maxillary measurement, statistically significant differences were verified for the molar and canine. At the mandibular level, statistically significant differences were only verified in the first molar. CONCLUSIONS: The Invisalign® clear aligners are effective for simultaneous intra-arch expansion in both jaws.

Luminal Rank loss decreases cell fitness leading to basal cell bipotency in parous mammary glands.

Rank signaling pathway regulates mammary gland homeostasis and epithelial cell differentiation. Although Rank receptor is expressed by basal cells and luminal progenitors, its role in each individual cell lineage remains unclear. By combining temporal/lineage specific Rank genetic deletion with lineage tracing techniques, we found that loss of luminal Rank reduces the luminal progenitor pool and leads to aberrant alveolar-like differentiation with high protein translation capacity in virgin mammary glands. These Rank-deleted luminal cells are unable to expand during the first pregnancy, leading to lactation failure and impairment of protein synthesis potential in the parous stage. The unfit parous Rank-deleted luminal cells in the alveoli are progressively replaced by Rank-proficient cells early during the second pregnancy, thereby restoring lactation. Transcriptomic analysis and functional assays point to the awakening of basal bipotency after pregnancy by the induction of Rank/NF-κB signaling in basal parous cell to restore lactation and tissue homeostasis.

Coronal Repercussions of the Maxillary Central Incisor Torque in the First Set of Aligners: A Retrospective Study.

Coronal torque is one of the key factors in orthodontic treatment. An adequate torque value has an impact on aesthetics and soft tissue profile. The aim of this quantitative, comparative and observational longitudinal cohort study was to analyze the efficacy of the maxillary central incisor coronal torque in the Invisalign® system and evaluate the relation between coronal torque movement and patient's facial biotype. In total, 27 patients were selected. The planned movements (TP) were obtained from the Invisalign Doctor Site® using mathematical formulas that consider the T0 measurements. Pre-treatment (T0) and after full use of the first set of aligners (T1) scanners were evaluated using Geomagic® Control X TM by superimposing T0 and T1 models using a transverse plane and the long axis of the tooth crown. IBM® SPSS® software was used for statistical purposes. We found statistically significant differences between T0 and T1 in pro-inclination and retro-inclination, as well as between achieved and planned values in pro-inclination (p = 0.011). We verified that hyperdivergent clinical cases presented higher mean values of coronal torque, and hypodivergent cases presented lower values. In pro-inclination, the differences between the planned and achieved values were greater in hypodivergent cases and smaller in hyperdivergent cases. In retro-inclination, the differences between the planned and achieved values were greater in normodivergent cases and smaller in hypodivergent cases. This study highlights that inefficacy is more accentuated in pro-inclination. Aligners are an effective tool for producing coronal repercussions of torque movement, being more effective in retro-inclination.

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration.

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.

Myocardial-specific R-spondin3 drives proliferation of the coronary stems primarily through the Leucine Rich Repeat G Protein coupled receptor LGR4.

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal R-spondin 3 receptor in the heart.

Genetic and Molecular Insights Into Genotype-Phenotype Relationships in Osteopathia Striata With Cranial Sclerosis (OSCS) Through the Analysis of Novel Mouse Wtx Mutant Alleles.

The X-linked WTX/AMER1 protein constitutes an important component of the ß-catenin destruction complex that can both enhance and suppress canonical ß-catenin signaling. Somatic mutations in WTX/AMER1 have been found in a proportion of the pediatric kidney cancer Wilms' tumor. By contrast, germline mutations cause the severe sclerosing bone dysplasia osteopathia striata congenita with cranial sclerosis (OSCS), a condition usually associated with fetal or perinatal lethality in male patients. Here we address the developmental and molecular function of WTX by generating two novel mouse alleles. We show that in addition to the previously reported skeletal abnormalities, loss of Wtx causes severe midline fusion defects including cleft palate and ectopic synostosis at the base of the skull. By contrast, deletion of the C-terminal part of the protein results in only mild developmental abnormalities permitting survival beyond birth. Adult analysis, however, revealed skeletal defects including changed skull morphology and an increased whole-body bone density, resembling a subgroup of male patients carrying a milder, survivable phenotype. Molecular analysis in vitro showed that while ß-catenin fails to co-immunoprecipitate with the truncated protein, partial recruitment appears to be achieved in an indirect manner using AXIN/AXIN2 as a molecular bridge. Taken together our analysis provides a novel model for WTX-caused bone diseases and explains on the molecular level how truncation mutations in this gene may retain some of WTX-protein functions. © 2018 American Society for Bone and Mineral Research.

Coronary Artery Formation Is Driven by Localized Expression of R-spondin3.

Coronary arteries are essential to support the heart with oxygen, and coronary heart disease is one of the leading causes of death worldwide. The coronary arteries form at highly stereotyped locations and are derived from the primitive vascular plexus of the heart. How coronary arteries are remodeled and the signaling molecules that govern this process are poorly understood. Here, we have identified the Wnt-signaling modulator Rspo3 as a crucial regulator of coronary artery formation in the developing heart. Rspo3 is specifically expressed around the coronary stems at critical time points in their development. Temporal ablation of Rspo3 at E11.5 leads to decreased ß-catenin signaling and a reduction in arterial-specific proliferation. As a result, the coronary stems are defective and the arterial tree does not form properly. These results identify a mechanism through which localized expression of RSPO3 induces proliferation of the coronary arteries at their stems and permits their formation.

The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce ß-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

The Angiocrine Factor Rspondin3 Is a Key Determinant of Liver Zonation.

Liver zonation, the spatial separation of different metabolic pathways along the liver sinusoids, is fundamental for proper functioning of this organ, and its disruption can lead to the development of metabolic disorders such as hyperammonemia. Metabolic zonation involves the induction of ß-catenin signaling around the central veins, but how this patterned activity is established and maintained is unclear. Here, we show that the signaling molecule Rspondin3 is specifically expressed within the endothelial compartment of the central vein. Conditional deletion of Rspo3 in mice disrupts activation of central fate, demonstrating its crucial role in determining and maintaining ß-catenin-dependent zonation. Moreover, ectopic expression of Rspo1, a close family member of Rspo3, induces the expression of pericentral markers, demonstrating Rspondins to be sufficient to imprint a more central fate. Thus, Rspo3 is a key angiocrine factor that controls metabolic zonation of liver hepatocytes.

Characterization of human NLZ1/ZNF703 identifies conserved domains essential for proper subcellular localization and transcriptional repression.

NET family members have recently emerged as important players in the development of multiple structures, from the trachea of fly larvae to the vertebrate eye and human breast cancers. However, their mechanisms of action are still poorly understood, and we lack a detailed characterization of their functional domains, as well as gene expression patterns-particularly in adult mammals. Here, we present a characterization of human NLZ1/ZNF703 (NocA-like zinc finger 1/Zinc finger 703), one of the two human NET family member genes. We show that the gene is ubiquitously expressed in adult human and mouse tissues, that three mRNA species with the same coding sequence are generated by alternative polyadenylation, and that the encoded protein contains six evolutionarily conserved domains, three of which are specific to NET proteins. Finally, we present functional evidence that these domains are necessary for proper subcellular distribution of and transcription repression by the NLZ1 protein, but not for its interaction with Groucho family co-repressors.

BRCA1 deficiency in skin epidermis leads to selective loss of hair follicle stem cells and their progeny.

The accurate maintenance of genomic integrity is essential for tissue homeostasis. Deregulation of this process leads to cancer and aging. BRCA1 is a critical mediator of this process. Here, we performed conditional deletion of Brca1 during epidermal development and found that BRCA1 is specifically required for hair follicle (HF) formation and for development of adult HF stem cells (SCs). Mice deficient for Brca1 in the epidermis are hairless and display a reduced number of HFs that degenerate progressively. Surprisingly, the interfollicular epidermis and the sebaceous glands remain unaffected by Brca1 deletion. Interestingly, HF matrix transient amplifying progenitors present increased DNA damage, p53 stabilization, and caspase-dependent apoptosis compared with the interfollicular and sebaceous progenitors, leading to hyperproliferation, apoptosis, and subsequent depletion of the prospective adult HF SCs. Concomitant deletion of p53 and Brca1 rescues the defect of HF morphogenesis and loss of HF SCs. During adult homeostasis, BRCA1 is dispensable for quiescent bulge SCs, but upon their activation during HF regeneration, Brca1 deletion causes apoptosis and depletion of Brca1-deficient bulge SCs. Our data reveal a major difference in the requirement of BRCA1 between different types of epidermal SCs and progenitors and during the different activation stages of adult HF SCs.

Identification of stem cell populations in sweat glands and ducts reveals roles in homeostasis and wound repair.

Sweat glands are abundant in the body and essential for thermoregulation. Like mammary glands, they originate from epidermal progenitors. However, they display few signs of cellular turnover, and whether they have stem cells and tissue-regenerative capacity remains largely unexplored. Using lineage tracing, we here identify in sweat ducts multipotent progenitors that transition to unipotency after developing the sweat gland. In characterizing four adult stem cell populations of glandular skin, we show that they display distinct regenerative capabilities and remain unipotent when healing epidermal, myoepithelial-specific, and lumenal-specific injuries. We devise purification schemes and isolate and transcriptionally profile progenitors. Exploiting molecular differences between sweat and mammary glands, we show that only some progenitors regain multipotency to produce de novo ductal and glandular structures, but that these can retain their identity even within certain foreign microenvironments. Our findings provide insight into glandular stem cells and a framework for the further study of sweat gland biology.

mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma.

CONTEXT: There are several genetic and molecular evidences suggesting dysregulation of the mammalian target of rapamycin (mTOR) pathway in thyroid neoplasia. Activation of the phosphatidylinositol-3-kinase/AKT pathway by RET/PTC and mutant RAS has already been demonstrated, but no data have been reported for the BRAF(V600E) mutation. OBJECTIVE: The aim of this study was to evaluate the activation pattern of the mTOR pathway in malignant thyroid lesions and whether it may be correlated with known genetic alterations, as well as to explore the mechanisms underlying mTOR pathway activation in these neoplasias. RESULTS: We observed, by immunohistochemical evaluation, an up-regulation/activation of the mTOR pathway proteins in thyroid cancer, particularly in conventional papillary thyroid carcinoma (cPTC). Overactivation of the mTOR signaling was particularly evident in cPTC samples harboring the BRAF(V600E) mutation. Transfection assays with BRAF expression vectors as well as BRAF knockdown by small interfering RNA revealed a positive association between BRAF expression and mTOR pathway activation, which appears to be mediated by pLKB1 Ser428, and emerged as a possible mechanism contributing to the association between BRAF mutation and mTOR pathway up-regulation. When we evaluated the rapamycin in the growth of thyroid cancer cell lines, we detected that cell lines with activating mutations in the MAPK pathway show a higher sensitivity to this drug. CONCLUSIONS: We determined that the AKT/mTOR pathway is particularly overactivated in human cPTC harboring the BRAF(V600E) mutation. Moreover, our results suggest that the mTOR pathway could be a good target to enhance therapy effects in certain types of thyroid carcinoma, namely in those harboring the BRAF(V600E) mutation.

Distinct stem cells contribute to mammary gland development and maintenance.

The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

Evaluation of the mTOR pathway in ocular (uvea and conjunctiva) melanoma.

Ocular melanoma is the most common eye malignancy in adults. It usually arises in the uvea, mostly in the choroid and less frequently in the conjunctiva. There is no curative therapy available when it becomes metastatic. The etiopathogenesis of uvea and conjunctiva melanomas is still poorly understood. The mammalian target of rapamycin (mTOR) pathway is involved in many biological processes and has been implicated in the development of cutaneous melanoma tumours. The mTOR pathway is an important target for anticancer drug development, and an inhibitor of this pathway has already been approved for use in humans to treat advanced renal cell carcinoma. The aim of this study was to evaluate the contribution of the mTOR pathway in uvea and conjunctiva melanomas. We analysed specific mTOR pathway effectors using immunohistochemical analysis of 30 uvea and eight conjunctiva melanoma samples. We assessed the association with prognostic clinical-pathological features, and performed mutational analysis on the BRAF and NRAS genes. None of the cases had mutations in either BRAF or NRAS. Expression of phospho-AKT Thr308 was associated with metastatic uvea melanomas. In conjunctiva melanomas, overactivation of the mTOR pathway, as confirmed by high phospho-AKT Ser473 and Thr308, S6 and p4EBP1 Thr37/46 levels, was associated with adverse prognostic parameters (mitotic index and tumour thickness). Conjunctiva melanomas displayed high expression of phospho-mTOR effectors in contrast with uvea melanomas, in which PTEN seemed to downregulate the mTOR pathway. Characterizing the expression of PTEN, AKT and pS6 Ser235/236 might be a useful predictive tool for deciding whether to use mTOR inhibitors to treat conjunctiva melanomas.

Conventional and molecular cytogenetics of human non-medullary thyroid carcinoma: characterization of eight cell line models and review of the literature on clinical samples.

BACKGROUND: Cell lines are often poorly characterized from a genetic point of view, reducing their usefulness as tumor models. Our purpose was to assess the genetic background of eight commonly used human thyroid carcinoma models and to compare the findings with those reported for primary tumors of the gland. METHODS: We used chromosome banding analysis and comparative genomic hybridization to profile eight non-medullary thyroid carcinoma cell lines of papillary (TPC-1, FB2, K1 and B-CPAP), follicular (XTC-1) or anaplastic origin (8505C, C643 and HTH74). To assess the representativeness of the findings, we additionally performed a thorough review of cytogenetic (n = 125) and DNA copy number information (n = 270) available in the literature on clinical samples of thyroid carcinoma. RESULTS: The detailed characterization of chromosomal markers specific for each cell line revealed two cases of mistaken identities: FB2 was shown to derive from TPC-1 cells, whereas K1 cells have their origin in cell line GLAG-66. All cellular models displayed genomic aberrations of varying complexity, and recurrent gains at 5p, 5q, 8q, and 20q (6/7 cell lines) and losses at 8p, 13q, 18q, and Xp (4/7 cell lines) were seen. Importantly, the genomic profiles were compatible with those of the respective primary tumors, as seen in the meta-analysis of the existing literature data. CONCLUSION: We provide the genomic background of seven independent thyroid carcinoma models representative of the clinical tumors of the corresponding histotypes, and highlight regions of recurrent aberrations that may guide future studies aimed at identifying target genes. Our findings further support the importance of routinely performing cytogenetic studies on cell lines, to detect cross-contamination mishaps such as those identified here.

Cyclic AMP inhibits the proliferation of thyroid carcinoma cell lines through regulation of CDK4 phosphorylation.

How cyclic AMP (cAMP) could positively or negatively regulate G1 phase progression in different cell types or in cancer cells versus normal differentiated counterparts has remained an intriguing question for decades. At variance with the cAMP-dependent mitogenesis of normal thyroid epithelial cells, we show here that cAMP and cAMP-dependent protein kinase activation inhibit S-phase entry in four thyroid carcinoma cell lines that harbor a permanent activation of the Raf/ERK pathway by different oncogenes. Only in Ret/PTC1-positive TPC-1 cells did cAMP markedly inhibit the Raf/ERK cascade, leading to mTOR pathway inhibition, repression of cyclin D1 and p21 and p27 accumulation. p27 knockdown did not prevent the DNA synthesis inhibition. In the other cells, cAMP little affected these signaling cascades and levels of cyclins D or CDK inhibitors. However, cAMP differentially inhibited the pRb-kinase activity and T172-phosphorylation of CDK4 complexed to cyclin D1 or cyclin D3, whereas CDK-activating kinase activity remained unaffected. At variance with current conceptions, our studies in thyroid carcinoma cell lines and previously in normal thyrocytes identify the activating phosphorylation of CDK4 as a common target of opposite cell cycle regulations by cAMP, irrespective of its impact on classical mitogenic signaling cascades and expression of CDK4 regulatory partners.

Intragenic mutations in thyroid cancer.

The close genotype-phenotype relationship that characterizes thyroid oncology stimulated the authors to address this article by using a mixed, genetic and phenotypic approach. As such, this article addresses the following aspects of intragenic mutations in thyroid cancer: thyroid stimulating hormone receptor and guanine-nucleotide-binding proteins of the stimulatory family mutations in hyperfunctioning tumors; mutations in RAS and other genes and aneuploidy; PAX8-PPARgamma rearrangements; BRAF mutations; mutations in oxidative phosphorylation and Krebs cycle genes in Hürthle cell tumors; mutations in succinate dehydrogenase genes in medullary carcinoma and C-cell hyperplasia; and mutations in TP53 and other genes in poorly differentiated and anaplastic carcinomas.

Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines.

OBJECTIVE: Our aim was to characterize the molecular and genotypic profile of eight thyroid carcinoma-derived cell lines-TPC1, FB2, B-CPAP, K1, XTC-1, C643, 8505C, and Hth74-in order to use them as in vitro models of thyroid carcinogenesis. DESIGN: We evaluated the expression of five thyroid-specific genes (Tg, TSHr, TPO, PAX8, and TTF-1) to establish the cell lineage and to assess the differentiation status of each of the cell lines. We screened for mutations in the most relevant oncogenes/tumor suppressor genes affected in thyroid carcinogenesis: RAS, BRAF, CTNNB1, and TP53 along with RET/PTC rearrangements. Considering the putative relevance in general carcinogenesis, we have also studied other molecules such as EGFR, PI3K, RAF-1, and THRB. To determine the genetic identity of the cell lines, we performed genotypic analysis. MAIN OUTCOME: The panel of cell lines we have studied displayed activation of several oncogenes (BRAF, RAS, RET/PTC) and inactivation of tumor suppressor genes (TP53) known to be important for thyroid carcinogenesis. Two of the cell lines-TPC1 and FB2-shared the same genotypic profile, probably representing clones of an ancestor cell line (TPC1). CONCLUSION: Due to their different molecular alterations, these cell lines represent a valuable tool to study the molecular mechanisms underlying thyroid carcinogenesis. We suggest that genotypic analyses should be included as a routine procedure to guarantee the uniqueness of each cell line used in research.

Thyroid hormone receptor beta mutations in the 'hot-spot region' are rare events in thyroid carcinomas.

Thyroid cancer constitutes the most frequent endocrine neoplasia. Targeted expression of rearranged during transfection (RET)/papillary thyroid carcinoma (PTC) and V600E V-raf murine sarcoma viral oncogene homolog B1 (BRAF) to the thyroid glands of transgenic mice results in tumours similar to those of human PTC, providing evidence for the involvement of these oncogenes in PTC. Kato et al. developed a mouse model that mimics the full spectrum of the human follicular form of thyroid cancer (FTC). FTC rapidly develops in these mice through introduction of the thyroid hormone receptor beta (THRB)(PV) mutant on the background of the inactivated THRB wt locus. Our aim was to verify if, in the context of human follicular thyroid carcinogenesis, THRB acted as a tumour suppressor gene. We screened for mutations of the THRB gene in the hot-spot region, spanning exons 7-10, in 51 thyroid tumours and six thyroid cancer cell lines by PCR and direct sequencing. We did not find mutations in any of the tumours or cell lines analysed. Our findings suggest that, in contrast to the findings on the THRB-mutant transgenic mice, THRB gene mutations are not a relevant mechanism for human thyroid carcinogenesis.