Reptile enamel matrix proteins: Selection, divergence, and functional constraint

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.We calculated the percent identity between amino acid sequences of ameloblastin from various reptile groups. Crocodilians exhibit the highest sequence identity, while identity across squamates was substantially lower. Upon closer examination of the individual squamate clades, however, we found that identity values are actually much higher in snakes, with much of the variation existing between the various lizard infraorders.HIGHLIGHTSReptile enamel matrix proteins are under moderate purifying selection despite polyphyodonty and simple enamel structure.Sequence identity in reptile enamel matrix proteins exhibit correlation with divergence times in spite of differences in substitution rates.Reptile amelogenin operates under a distinct selection regime compared with ameloblastin and enamelin.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/1/jezb22857.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/2/jezb22857-sup-0001-Supplementary_file.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/3/jezb22857-sup-0007-Supplementary_file_S8-DAMBE-Saturation.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/4/jezb22857-sup-0002-Supplementary_file_S1-SpeciesTable.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/5/jezb22857-sup-0003-Supplementary_file_S2_Alignments.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/6/jezb22857-sup-0008-Supplementary_File_S9.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/7/jezb22857-sup-0005-Supplementary_file_S6.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/8/jezb22857_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/9/jezb22857-sup-0009-Supplementary_file_Reptiles.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150577/10/jezb22857-sup-0006-Supplementary_file_S7-DIVERGE.pd

European clinical guidelines for Tourette syndrome and other tic disorders—version 2.0. Part III: pharmacological treatment

In 2011, the European Society for the Study of Tourette Syndrome (ESSTS) published the first European guidelines for Tourette Syndrome (TS). We now present an update of the part on pharmacological treatment, based on a review of new literature with special attention to other evidence-based guidelines, meta-analyses, and randomized double-blinded studies. Moreover, our revision took into consideration results of a recent survey on treatment preferences conducted among ESSTS experts. The first preference should be given to psychoeducation and to behavioral approaches, as it strengthens the patients’ self-regulatory control and thus his/her autonomy. Because behavioral approaches are not effective, available, or feasible in all patients, in a substantial number of patients pharmacological treatment is indicated, alone or in combination with behavioral therapy. The largest amount of evidence supports the use of dopamine blocking agents, preferably aripiprazole because of a more favorable profile of adverse events than first- and second-generation antipsychotics. Other agents that can be considered include tiapride, risperidone, and especially in case of co-existing attention deficit hyperactivity disorder (ADHD), clonidine and guanfacine. This view is supported by the results of our survey on medication preference among members of ESSTS, in which aripiprazole was indicated as the drug of first choice both in children and adults. In treatment resistant cases, treatment with agents with either a limited evidence base or risk of extrapyramidal adverse effects might be considered, including pimozide, haloperidol, topiramate, cannabis-based agents, and botulinum toxin injections. Overall, treatment of TS should be individualized, and decisions based on the patient’s needs and preferences, presence of co-existing conditions, latest scientific findings as well as on the physician’s preferences, experience, and local regulatory requirements

Mineral maturity and crystallinity index are distinct characteristics of bone mineral

The purpose of this study was to test the hypothesis that mineral maturity and crystallinity index are two different characteristics of bone mineral. To this end, Fourier transform infrared microspectroscopy (FTIRM) was used. To test our hypothesis, synthetic apatites and human bone samples were used for the validation of the two parameters using FTIRM. Iliac crest samples from seven human controls and two with skeletal fluorosis were analyzed at the bone structural unit (BSU) level by FTIRM on sections 2–4 lm thick. Mineral maturity and crystallinity index were highly correlated in synthetic apatites but poorly correlated in normal human bone. In skeletal fluorosis, crystallinity index was increased and maturity decreased, supporting the fact of separate measurement of these two parameters. Moreover, results obtained in fluorosis suggested that mineral characteristics can be modified independently of bone remodeling. In conclusion, mineral maturity and crystallinity index are two different parameters measured separately by FTIRM and offering new perspectives to assess bone mineral traits in osteoporosis

Remodeling the Proteostasis Network to Rescue Glucocerebrosidase Variants by Inhibiting ER-Associated Degradation and Enhancing ER Folding

Gaucher’s disease (GD) is characterized by loss of lysosomal glucocerebrosidase (GC) activity. Mutations in the gene encoding GC destabilize the protein’s native folding leading to ER-associated degradation (ERAD) of the misfolded enzyme. Enhancing the cellular folding capacity by remodeling the proteostasis network promotes native folding and lysosomal activity of mutated GC variants. However, proteostasis modulators reported so far, including ERAD inhibitors, trigger cellular stress and lead to induction of apoptosis. We show herein that lacidipine, an L-type Ca2+ channel blocker that also inhibits ryanodine receptors on the ER membrane, enhances folding, trafficking and lysosomal activity of the most severely destabilized GC variant achieved via ERAD inhibition in fibroblasts derived from patients with GD. Interestingly, reprogramming the proteostasis network by combining modulation of Ca2+ homeostasis and ERAD inhibition remodels the unfolded protein response and dramatically lowers apoptosis induction typically associated with ERAD inhibition

Empirical Evaluation of Bone Extraction Protocols

The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone

Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

Abstract BACKGROUND: Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2(-/-) mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts. METHODOLOGY/PRINCIPAL FINDINGS: Alkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix. CONCLUSIONS/SIGNIFICANCE: Together with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis

Effects of THBS3, SPARC and SPP1 expression on biological behavior and survival in patients with osteosarcoma

BACKGROUND: Osteosarcoma is a very aggressive tumor with a propensity to metastasize and invade surrounding tissue. Identification of the molecular determinants of invasion and metastatic potential may guide the development of a rational strategy for devising specific therapies that target the pathways leading to osteosarcoma. METHODS: In this study, we used pathway-focused low density expression cDNA arrays to screen for candidate genes related to tumor progression. Expression patterns of the selected genes were validated by real time PCR on osteosarcoma patient tumor samples and correlated with clinical and pathological data. RESULTS: THBS3, SPARC and SPP1 were identified as genes differentially expressed in osteosarcoma. In particular, THBS3 was expressed at significantly high levels (p = 0.0001) in biopsies from patients with metastasis at diagnosis, which is a predictor of worse overall survival, event-free survival and relapse free survival at diagnosis. After chemotherapy, patients with tumors over-expressing THBS3 have worse relapse free survival. High SPARC expression was found in 51/55 (96.3%) osteosarcoma samples derived from 43 patients, and correlated with the worst event-free survival (p = 0.03) and relapse free survival (p = 0.07). Overexpression of SPP1 was found in 47 of 53 (89%) osteosarcomas correlating with better overall survival, event-free survival and relapse free survival at diagnosis. CONCLUSION: In this study three genes were identified with pattern of differential gene expression associated with a phenotypic role in metastasis and invasion. Interestingly all encode for proteins involved in extracellular remodeling suggesting potential roles in osteosarcoma progression. This is the first report on the THBS3 gene working as a stimulator of tumor progression. Higher levels of THBS3 maintain the capacity of angiogenesis. High levels of SPARC are not required for tumor progression but are necessary for tumor growth and maintenance. SPP1 is not necessary for tumor progression in osteosarcoma and may be associated with inflammatory response and bone remodeling, functioning as a good biomarker