Evaluation of results after 112 radioembolizations with 90Y-microspheres.

AIM: To determine the results of radioembolization transarterial (TARE), in the treatment of liver tumors, a retrospective evaluation was performed after 112 TARE with 90Y-microspheres administered in 82 patients in a single hospital, analyzing efficacy and safety, after a follow-up greater than or equal to 1 year post-TARE in all patients, and evaluating the possible relationship between treatment response and patient survival. MATERIAL AND METHODS: We have administered 57 single TARE and 55 multiple TARE in patients with hepatocellular carcinoma (53), liver metastases (25) and cholangiocarcinoma (4), with prior multidisciplinary evaluation, clinical, angiographic and gammagraphic (planar/SPECT/SPECT-CT with 99mTc-MAA), multicompartment model (MIRD equations), post-TARE screening (planar/SPECT/SPECT-CT), clinical and radiological follow-up, tumor response evaluation (mRECIST criteria) and Kaplan-Meier analysis to determine progression-free survival (PFS) and overall survival (OS). RESULTS: Therapeutic intention was palliative (82%) and as bridge to liver transplantation/surgical resection (17%). We obtained response (R), complete or partial, in 65.9% of cases. One year after TARE 34.7% of patients with R and 19.2% of non-R were progression-free (P: .003), with OS of 80% for R and 37.5% for non-R (P: .001). Survival analysis showed median OS of 18 months (95% CI 15.7-20.3) for R and 9 months (95% CI 6.1-11.8) for non-R (P: .03). We found mild (27.6%) and severe (5.3%) side effects, all of them resolved, without higher incidence after multiple TARE. CONCLUSION: TARE with 90Y-microspheres, in appropriately selected patients with liver tumors, provides therapeutic efficacy and low rate of toxicity, with higher PFS and OS in patients with TARE response compared to those who did not respond.

The freezing method of cleavage stage embryos has no impact on the weight of the newborns.

PURPOSE: The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification. METHODS: This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study. RESULTS: There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4%). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0%). The live birth rates were similar (19.5 vs 19.1%) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9. CONCLUSIONS: The freezing method has no impact on the weight of the newborn. With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

RAS mutations and cetuximab in locally advanced rectal cancer: results of the EXPERT-C trial.

BACKGROUND: RAS mutations predict resistance to anti-epidermal growthfactor receptor (EGFR) monoclonal antibodies in metastatic colorectal cancer. We analysed RAS mutations in 30 non-metastatic rectal cancer patients treated with or without cetuximab within the 31 EXPERT-C trial. METHODS: Ninety of 149 patients with tumours available for analysis were KRAS/BRAF wild-type, and randomly assigned to capecitabine plus oxaliplatin (CAPOX) followed by chemoradiotherapy, surgery and adjuvant CAPOX or the same regimen plus cetuximab (CAPOX-C). Of these, four had a mutation of NRAS exon 3, and 84 were retrospectively analysed for additional KRAS (exon 4) and NRAS (exons 2/4) mutations by using bi-directional Sanger sequencing. The effect of cetuximab on study end-points in the RAS wild-type population was analysed. RESULTS: Eleven (13%) of 84 patients initially classified as KRAS/BRAF wild-type were found to have a mutation in KRAS exon 4 (11%) or NRAS exons 2/4 (2%). Overall, 78/149 (52%) assessable patients were RAS wild-type (CAPOX, n=40; CAPOX-C, n=38). In this population, after a median follow-up of 63.8months, in line with the initial analysis, the addition of cetuximab was associated with numerically higher, but not statistically significant, rates of complete response (15.8% versus 7.5%, p=0.31), 5-year progression-free survival (75.5% versus 67.5%, hazard ratio (HR) 0.61, p=0.25) and 5-year overall survival (83.8% versus 70%, HR 0.54, p=0.20). CONCLUSIONS: RAS mutations beyond KRAS exon 2 and 3 were identified in 17% of locally advanced rectal cancer patients. Given the small sample size, no definitive conclusions on the effect of additional RAS mutations on cetuximab treatment in this setting can be drawn and further investigation of RAS in larger studies is warranted.

Lobar lung resection in elderly patients with non-small cell lung carcinoma: impact of cardiac comorbidity on surgical outcome.

PRINCIPLES: The aim of this study was to evaluate the impact of cardiac comorbidity on the perioperative morbidity and mortality after lobar lung resection for lung cancer in patients aged 70 years and older. METHODS: The medical records of all 68 patients ≥70 years, who underwent lobar lung resection for non-small cell lung cancer (NSCLC) from 2003 to 2011 at our department, were reviewed retrospectively. Twenty-two patients with a mean age of 76.3 years had cardiac comorbidities (Group A) including previous cardiac operations in 4 patients, previous myocardial infarction in 5 patients, previous coronary stent insertion in 3 patients, medically treated coronary artery disease in 10 patients and medically treated valvular heart disease in 2 patients whereas 46 patients (mean age = 74.5 years) had no previous cardiac history (Group B). RESULTS: There were no significant differences in postoperative morbidity (13.6% in Group A vs. 17.4% in Group B) between both groups. No in-hospital mortality was observed in both groups. CONCLUSION: In our experience lobar lung resections for NSCLC in elderly patients with cardiac comorbidity seem to be a safe therapy option for this increasing subpopulation. Though, our retrospective data with the small number of study objects require further confirmation in larger prospective trials.

[Breast cancer metastases in the iris: does treatment modifies natural history of the illness?]. / Metástasis en iris de un carcinoma de mama: ¿modifica el tratamiento la historia natural de la enfermedad?

The 4-5% of the breast cancer patients have metastases in the eye. We present the case of a 30-year-old woman with an infiltrant duct carcinoma of the breast pT2N2M0 HER2 positive. Six months after primary radical treatment she had a systemic relapse with multiples metastatic sites, so several treatment with trastuzumab in combination with chemotherapy were started. After 4 years patient presented multiple white-coloured micronodules in the iris of the right eye. Only a 3-7.8% of ocular metastases are located in the iris. With mantenaince therapy with trastuzumab natural history of the illness has changed. Several studies had analyzed if metastases in the brain during treatment with trastuzumab have increased in comparison with the pretrastuzumab era. The infrequent presentation of metastases in the anterior uveal makes difficult to establish if it is an spontaneous fact or if it is favoured by trastuzumab treatment.

Characterization of the sec1-1 and sec1-11 mutations.

Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation. To better understand the function of Sec1 proteins we have identified the nature of the temperature-sensitive mutations in sec1-1 and sec1-11. The sec1-1 mutation changes a conserved glycine(443) to glutamic acid. The sec1-11 mutation changes a highly conserved arginine(432) to proline. Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p. Compared to the wild-type Sec1p the mutant proteins are less abundant even at the permissive temperature. Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation. The remaining part is sufficient for growth and protein secretion at 24 degrees C and thus is likely to be properly folded. At 37 degrees C the mutant proteins become non-functional. In pulse-chase-type experiments the newly synthesized Sec1-1 and Sec1-11 proteins decayed similarly with the wild-type protein. Thus, the non-functionality of the mutant proteins cannot be explained by denaturation-induced degradation only. It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins. The mutant proteins were unable to interact with the Sec1p-interacting proteins Mso1p and Sso2p in the two-hybrid assay, even at the permissive temperature. These results localize sec1-1 and sec1-11 mutations to a domain of Sec1p and suggest a mechanism by which sec1-1 and sec1-11 cells become temperature-sensitive.

Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae.

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes.

Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation.

Odontoblasts differentiate from the cells of the dental papilla, and it has been well-established that their differentiation in developing teeth is induced by the dental epithelium. In experimental studies, no other mesenchymal cells have been shown to have the capacity to differentiate into odontoblasts, indicating that the dental papilla cells have been committed to odontoblast cell lineage during earlier developmental stages. We propose that the advancing differentiation within the odontoblast cell lineage is regulated by sequential epithelial signals. The first epithelial signals from the early oral ectoderm induce the odontogenic potential in the cranial neural crest cells. The next step in the determination of the odontogenic cell lineage is the development of the dental papilla from odontogenic mesenchyme. The formation of the dental papilla starts at the onset of the transition from the bud to the cap stage of tooth morphogenesis, and this is regulated by epithelial signals from the primary enamel knot. The primary enamel knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes fully developed and morphologically discernible in the cap-stage dental epithelium and expresses at least ten different signaling molecules belonging to the BMP, FGF, Hh, and Wnt families. In molar teeth, secondary enamel knots appear in the enamel epithelium at the sites of the future cusps. They also express several signaling molecules, and their formation precedes the folding and growth of the epithelium. The differentiation of odontoblasts always starts from the tips of the cusps, and therefore, it is conceivable that some of the signals expressed in the enamel knots may act as inducers of odontoblast differentiation. The functions of the different signals in enamel knots are not precisely known. We have shown that FGFs stimulate the proliferation of mesenchymal as well as epithelial cells, and they may also regulate the growth of the cusps. We have proposed that the enamel knot signals also have important roles, together with mesenchymal signals, in regulating the patterning of the cusps and hence the shape of the tooth crown. We suggest that the enamel knots are central regulators of tooth development, since they link cell differentiation to morphogenesis.

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.

Evolutionary modification of development in mammalian teeth: quantifying gene expression patterns and topography.

The study of mammalian evolution often relies on detailed analysis of dental morphology. For molecular patterning to play a role in dental evolution, gene expression differences should be linkable to corresponding morphological differences. Because teeth, like many other structures, are complex and evolution of new shapes usually involves subtle changes, we have developed topographic methods by using Geographic Information Systems. We investigated how genetic markers for epithelial signaling centers known as enamel knots are associated with evolutionary divergence of molar teeth in two rodent species, mouse and vole. Our analysis of expression patterns of Fgf4, Lef1, p21, and Shh genes in relation to digital elevation models of developing tooth shapes shows that molecular prepatterns predict the lateral cusp topography more than a day in advance. A heterotopic shift in the molecular prepatterns can be implicated in the evolution of mouse molar, changing locations from which historically homologous cusps form. The subtle but measurable heterotopic shifts may play a large role in the evolution of tooth cusp topographies. However, evolutionary increase in the number of longitudinal cusps in vole molar has involved accelerated longitudinal growth and iterative addition of new cusps without changes in lateral cusp topography. The iterative addition of cusps after the establishment of lateral cusp topography may limit the independence of individual morphological features used in evolutionary studies. The diversity of mammalian molar patterns may largely result from the heterotopic and iterative processes.

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

Munc18-2, a functional partner of syntaxin 3, controls apical membrane trafficking in epithelial cells.

The Sec1-related proteins bind to syntaxin family t-SNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.

Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast.

Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

Treatment of subcapital fractures of the fifth metacarpal bone: a prospective randomised comparison between functional treatment and reposition and splinting.

We did a prospective study to compare the results of treatment of subcapital fractures of the fifth metacarpal bone by closed reduction and splinting or by functional treatment. Twenty-nine consecutive patients were randomly divided into the two treatment groups (functional n = 14, and reposition and splinting n = 15). The results of treatment were satisfactory in both groups. Functionally treated patients recovered their grip force and range of movement of the affected hand a little sooner. All fractures in both groups had united within three months. There were no complications. We conclude that subcapital fractures of the fifth metacarpal bone can successfully be treated without closed reduction and splinting.

Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema regions.

Rodents have a toothless diastema region between the incisor and molar teeth which may contain rudimentary tooth germs. We found in upper diastema region of the mouse (Mus musculus) three small tooth germs which developed into early bud stage before their apoptotic removal, while the sibling vole (Microtus rossiaemeridionalis) had only a single but larger tooth germ in this region, and this developed into late bud stage before regressing apoptotically. To analyze the genetic mechanisms of the developmental arrest of the rudimentary tooth germs we compared the expression patterns of several developmental regulatory genes (Bmp2, Bmp4, Fgf4, Fgf8, Lef1, Msx1, Msx2, p21, Pitx2, Pax9 and Shh) between molars and diastema buds of mice and voles. In diastema tooth buds the expression of all the genes differed from that of molars. The gene expression patterns suggest that the odontogenic program consists of partially independent signaling cascades which define the exact location of the tooth germ, initiate epithelial budding, and transfer the odontogenic potential from the epithelium to the underlying mesenchyma. Although the diastema regions of the two species differed, in both species the earliest difference that we found was weaker expression of mesenchymal Pax9 in the diastema region than in molar and incisor regions at the dental lamina stage. However, based on earlier tissue recombination experiments it is conceivable that the developmental arrest is determined by the early oral epithelium.

DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability.

DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.

SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.

The exocyst is an essential multiprotein complex mediating polarized secretion in yeast. Here we describe a gene, SEM1, that can multicopy-suppress exocyst mutants sec3-2, sec8-9, sec10-2, and sec15-1. SEM1 is highly conserved among eukaryotic species. Its human homologue, DSS1, has been suggested as a candidate gene for the split hand/split foot malformation disorder. SEM1 is not an essential gene. However, its deletion rescued growth of the temperature-sensitive exocyst mutants sec3-2, sec8-9, sec10-1, and sec15-1 at the restrictive temperature. Cell fractionation showed that Sem1p is mainly cytosolic but also associates with the microsomal fraction. In linear sucrose gradients, Sem1p cosedimented with the exocyst component Sec8p. In diploid cells that normally do not form pseudohyphae (S288C background), deletion of SEM1 triggered pseudohyphal growth. This phenotype was abolished after reintroduction of either SEM1 or the mouse homologue Dss1 into the cells. In diploids that have normal capacity for pseudohyphal growth (Sigma1278b background), deletion of SEM1 enhanced filamentous growth. The functionality of both SEM1 and Dss1 in a differentiation process in yeast suggests that Dss1 indeed could be the gene affected in the split hand/split foot malformation disorder. These results characterize SEM1 as a regulator of both exocyst function and pseudohyphal differentiation and suggest a unique link between these two cellular functions in yeast.

Association of developmental regulatory genes with the development of different molar tooth shapes in two species of rodents.

While the evolutionary history of mammalian tooth shapes is well documented in the fossil record, the developmental basis of their tooth shape evolution is unknown. We investigated the expression patterns of eight developmental regulatory genes in two species of rodents with different molar morphologies (mouse, Mus musculus and sibling vole, Microtus rossiaemeridionalis). The genes Bmp-2, Bmp-4, Fgf-4 and Shh encode signal molecules, Lef-1, Msx-1 and Msx-2, are transcription factors and p21CIP1/WAF1 participates in the regulation of cell cycle. These genes are all known to be associated with developmental regulation in mouse molars. In this paper we show that the antisense mRNA probes made from mouse cDNA cross-hybridized with vole tissue. The comparisons of gene expression patterns and morphologies suggest that similar molecular cascades are used in the early budding of tooth germs, in the initiation of tooth crown base formation, and in the initiation of each cusp's development. Furthermore, the co-localization of several genes indicate that epithelial signalling centres function at the three stages of morphogenesis. The earliest signalling centre in the early budding epithelium has not been reported before, but the latter signalling centres, the primary and the secondary enamel knots, have been studied in mouse. The appearance of species-specific tooth shapes was manifested by the regulatory molecules expressed in the secondary enamel knots at the areas of future cusp tips, whilst the mesenchymal gene expression patterns had a buccal bias without similar species-specific associations.