Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice.

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.

Novel PAX9 gene polymorphisms and mutations and susceptibility to tooth agenesis in the Czech population.

OBJECTIVES: Tooth agenesis is one of the most common developmental anomalies in humans. Genetic and environmental factors may be of etiological importance in this condition. Among genes involved in tooth morphogenesis, mutations in PAX9, MSX1, AXIN2, WNT10a, and EDA genes have been associated with tooth agenesis. The aim of our study was to investigate the relationship between the PAX9 gene variants and tooth agenesis in the Czech population. METHODS: The selected regions of the PAX9 gene were analysed by direct sequencing and compared with the reference sequence from the GenBank online database (NCBI). RESULTS: We found several novel variants in the PAX9 gene, e.g. insertion g.5100_5101insC (rs11373281) with simultaneous substitution g.5272C>G (rs4904155) in exon 1, and mutation g.10934C>T (Gly203Gly, rs61754301) in exon 3. In subjects with full dentition we observed polymorphisms g.10276A>G (rs12882923) and g.10289A>G (rs12883049) in IVS2 (intervening sequence 2) previously related to tooth agenesis in Polish study. CONCLUSIONS: In our study we excluded a direct effect of rs12882923 and rs12883049 polymorphisms on the dental agenesis in the Czech population. All described PAX9 genetic variants were present both in patients with tooth agenesis and controls. We expect that tooth agenesis in our cohort of patients is caused by mutations in regions different from PAX9 exons analyzed in our study.

A screen of a large Czech cohort of oligodontia patients implicates a novel mutation in the PAX9 gene.

Tooth agenesis is one of the most common developmental anomalies in humans. To date, many mutations involving paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) genes have been identified. The aim of the present study was to perform screening for mutations and/or polymorphisms using the capillary sequencing method in the critical regions of PAX9 and MSX1 genes in a group of 270 individuals with tooth agenesis and in 30 healthy subjects of Czech origin. This screening revealed a previously unknown heterozygous g.9527G>T mutation in the PAX9 gene in monozygotic twins with oligodontia and three additional affected family members. The same variant was not found in healthy relatives. This mutation is located in intron 2, in the region recognized as the splice site between exon 2 and intron 2. We hypothesize that the error in pre-mRNA splicing may lead to lower expression of PAX9 protein and could have contributed to the development of tooth agenesis in the affected subjects.

Molecular mechanisms of neuropathological changes in Alzheimer's disease: a review.

More than 100 years after description of Alzheimer's disease (AD), two major pathological processes observed already by Alois Alzheimer, remain as the main explanation of the pathogenesis of Alzheimer's disease. Important molecular interactions leading to AD neuropathology were described in amyloid cascade and in tau protein function. No clinical trials with novel therapies based on amyloid cascade and tau protein hypotheses have been successful. The main aim of recent research is focused on the question what is primary mechanism leading to the molecular development of AD pathology. Promising explanation of triggering mechanism can be seen in vascular pathology that have direct influence on the development of pathological processes typical for Alzheimer disease. Novel insight into a number of cellular signaling mechanisms, as well as mitochondrial function in Alzheimer disease could also bring explanations of initial processes leading to the development of this pathology.

Interactive effect of MTHFR and ADRA2A gene polymorphisms on pathogenesis of schizophrenia.

OBJECTIVES: Increasing evidences support the importance of epigenetic control in schizophrenia pathogenesis. One of the enzymes involved in DNA methylation process through homocysteine metabolism is methylenetetrahydrofolate reductase (MTHFR). The most extensively studied variant in the MTHFR gene is the C677T polymorphism, resulting in reduced enzyme activity and elevated homocysteine level. METHODS: In sample of 192 schizophrenics and 213 healthy controls an increasing risk of schizophrenia associated with MTHFR 677 CT+TT genotype was found (OR=1.6, p=0.021). Association was also evaluated by considering the C677T polymorphism as an interaction with COMT Val158Met and ADRA2A C-1291G polymorphisms previously associated with schizophrenia risk using a logistic regression analysis. RESULTS: Previous studies of MTHFR*COMT (C677T*Val158Met) interaction in relation to schizophrenia resulted in inconsistent results. In our sample this interaction did not significantly differ between schizophrenics and control subjects. On the other hand analysis of MTHFR*ADRA2A (C677T*C-1291G) interaction revealed significant association between ADRA2A CC+CG genotype in the MTHFR TC+TT carriers (p=0.008). CONCLUSIONS: Our results support role of noradrenergic functions as well as previously proposed role of epigenetic control in the pathogenesis of schizophrenia. Further relevant studies including larger sample size and more markers are needed to prove our results.

Morphogenesis and bone integration of the mouse mandibular third molar.

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.

Comparative morphology of normal and cleft minipigs demonstrates dual origin of incisors.

OBJECTIVE: The incisors of the mammalian dental arch develop from tissues arising from separated facial prominences. These primordial craniofacial structures undergo complex morphogenetic processes as they merge and fuse in a time and space dependent fashion. However, local contributions of precursor facial prominences to the incisors that develop subsequently remain unknown. The purpose of this study was to characterize the development of all three deciduous upper rostral teeth in the pig (Sus scrofa f. domestica) for the identification of the likely facial prominence contributions to the incisors based on normal and pathological developmental relationships. DESIGN: Embryonic minipigs were collected between gestational days 20-36 (E20-36), processed for histological analysis and subjected to computerized 3D modelling. The location and morphology of the incisors (i) in these specimens were characterized and compared between developmental stages. A second set of neonatal minipigs displaying cleft lip and/or cleft palate defects were also obtained and incisor locations and eruption patterns were morphologically examined. RESULTS: Palate formation begins during the third week of gestation (E20) in the minipig with ossification of the premaxilla initiating soon afterwards (E24). The third incisor (i3) develops caudally to the contact seam formed by the fusion of the primary and secondary palates in normal embryos. All cleft animals displayed normal i3 and canine, on other hand, development of i1 and i2 was often disrupted similar to human. CONCLUSIONS: Our observations suggest a dual embryonic origin of the incisors in minipigs with the first and second incisors originating from the frontonasal prominence whilst the third incisor forms from tissues derived from the maxillary prominence.

Tooth-bone morphogenesis during postnatal stages of mouse first molar development.

The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0-2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex.

Early morphogenesis of heterodont dentition in minipigs.

The minipig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resemble that of humans. However, little information is available on the processes of tooth development in the pig. The purpose of this study was to classify the early stages of odontogenesis in minipigs from the initiation of deciduous dentition to the late bell stage when the successional dental lamina begins to develop. To analyze the initiation of teeth anlagens and the structural changes of dental lamina, a three-dimensional (3D) analysis was performed. At the earliest stage, 3D reconstruction revealed a continuous dental lamina along the length of the jaw. Later, the dental lamina exhibited remarkable differences in depth, and the interdental lamina was shorter. The dental lamina grew into the mesenchyme in the lingual direction, and its inclined growth was underlined by asymmetrical cell proliferation. After the primary tooth germ reached the late bell stage, the dental lamina began to disintegrate and fragmentize. Some cells disappeared during the process of lamina degradation, while others remained in small islands known as epithelial pearls. The minipig can therefore, inter alia, be used as a model organism to study the fate of epithelial pearls from their initiation to their contribution to pathological structures, primarily because of the clinical significance of these epithelial rests.

Molar tooth development in caspase-3 deficient mice.

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.

Proliferation and apoptosis in early molar morphogenesis-- voles as models in odontogenesis.

Proliferation and apoptosis play crucial roles in the development of multicellular organisms. Their precise balance is necessary for tissue homeostasis throughout life. The developing dentition is a suitable model to study proliferation and apoptosis during embryogenesis, but the corresponding studies have been carried out principally in the mouse. The present study aimed to examine proliferation and apoptosis in the vole (Microtus sp., Rodentia) during the early morphogenesis of the first upper molar and compare it to what is known from the mouse. To this end, apoptosis and proliferation were investigated using histology and computer-aided 3D reconstruction. Mitoses accumulated predominantly in the developing cervical loop. Apoptosis during early odontogenesis showed highly specific spatio-temporal patterns in the dental epithelium. Apoptotic bodies were localised in non-dividing cell populations. They accumulated in the same places as described in the mouse: antemolar vestiges (ED 12.5 15.5), enamel knot (ED 14.5 15.5), stalk and palatally along the whole first molar tooth germ longitudinal axis (ED 15 - 15.5). Early tooth development in the field vole, including the distribution of apoptosis and mitosis, is very similar to that reported in the mouse, with the exception of the antemolar region. The microtine antemolar vestige is preserved longer than the murine one. It is conceivable that additional distinct differences in morphogenetic processes appear later in tooth development.

Relationship between vestibular lamina, dental lamina, and the developing oral vestibule in the upper jaw of the field vole (Microtus agrestis, Rodentia).

Formation of the oral vestibule is ignored in most studies on tooth development, although dental and vestibular lamina are closely related to each other. Knowledge about morphogenetic processes shaping the oral vestibule is missing almost completely. The aim of this study was to assess the developmental relationship between dental and vestibular lamina as well as formation of the oral vestibule in the upper jaw of the field vole (Microtus agrestis), a small rodent representing an attractive model species for comparative dental studies. Three-dimensional reconstruction revealed that the upper vestibular lamina of the vole joins the antemolar part of the diastemal dental lamina, similar to mouse. Later, this lamina complex regresses and the vestibular lamina is separated from the molar epithelium. Participation of the vestibular lamina in dental lamina formation, as hypothesized for mouse, therefore remains unclear. Except for increased apoptosis in the regressing diastemal dental lamina, spatial segregation of mitoses or apoptoses could be detected neither in the jaw arch epithelium nor in the adjacent mesenchyme. Therefore, in contrast to tooth primordia, apoptosis and mitosis seem to play a minor role in shaping of the upper oral vestibule. The buccal vestibule develops secondarily, probably in consequence of general growth of the head and localized differentiation of cells.

Origin and developmental fate of vestigial tooth primordia in the upper diastema of the field vole (Microtus agrestis, Rodentia).

OBJECTIVE: Odontogenesis in voles is a convenient model to test hypotheses on tooth development generated from investigations in the mouse. Similar to other rodents, the functional dentition of the vole includes a toothless diastema. At its mesial end, a vestigial tooth bud has been found in the upper jaw of vole embryos. The aim of this study was to analyse the developmental dynamics of vestigial tooth structures in the upper diastema of the field vole and to compare it with the situation in the mouse. DESIGN: The development of odontogenic structures in the upper diastema of the field vole was investigated using serial histological sections and three-dimensional (3D) computer-aided reconstruction. RESULTS: A transient continuous dental lamina in the upper diastema of the field vole extended mesially to the first molar primordium, but was not continuous with the dental lamina in the incisor region. At its mesial limit, a large vestigial tooth primordium was regularly present. A further distinct vestigial bud was located mesially to the first molar primordium. The segmentation of the dental lamina suggested a potential to give rise to further vestiges in the upper diastema of the vole. CONCLUSIONS: In the prospective diastema of the vole exists as in the mouse a continuous dental lamina. Beside the prominent vestigial tooth bud in the mesial diastema, a further large bud was transiently located in front of the molars. The incorporation of dental epithelium into the first upper molar (M(1)) primordium in the vole differs from that in the mouse.

Heart development in the spotted dolphin (Stenella attenuata).

Marine mammals show many deviations from typical mammalian characteristics due to their high degree of specialization to the aquatic environment. In Cetaceans, some of the features of limbs and dentition resemble very ancestral patterns. In some species, hearts with a clearly bifid apex (a feature normally present during mammalian embryogenesis prior to completion of ventricular septation) have been described. However, there is a scant amount of data regarding heart development in Cetaceans, and it is not clear whether the bifid apex is the rule or the exception. We examined samples from a unique collection of embryonic dolphin specimens macroscopically and histologically to learn more about normal cardiac development in the spotted dolphin. It was found that during the dolphin's 280 days of gestation, the heart completes septation at about 35 days. However, substantial trabecular compaction, which normally occurs in chicks, mice, and humans at around that time period, was delayed until day 60, when coronary circulation became established. At that time, the apex still appeared bifid, similarly to early fetal mouse or rat hearts. By day 80, however, the heart gained a compacted, characteristic shape, with a single apex. It thus appears that the bifid apex in the adult Cetacean heart is probably particular to certain species, and its significance remains unclear.

Three-dimensional reconstruction studies and morphometric analysis of rudimental tooth primordia in the upper incisor region of the sheep (Ovis aries, Ruminantia).

The functional dentition of the domestic sheep lacks all upper incisors and the upper canines. Nevertheless, occurrence of a dental lamina and rudimental tooth primordia had been described in the upper incisor region of the sheep. The aim of this study was to describe temporo-spatial pattern of origin and regression of these rudimental tooth primordia by light microscopy, computer-aided three-dimensional reconstruction and morphometry of the dental epithelium. Transient existence of a dental lamina in the upper incisor region of the sheep and three epithelial thickenings on its deep mesenchymal margin has been observed at day of ontogeny (DO) 48-53. They could not been identified as full-value tooth primordia, because they did not induce differentiation of tooth mesenchyme, but they could represent last remnants of functional upper incisors in early ancestors of ruminants. Additionally, a large rudimental upper canine primordium near the sutura maxilloincisiva occurred at DO43, reached early cap stage at DO52 and started to regress at DO53. Thus, our findings showed a discrepancy between the embryonic and adult dental pattern in the sheep. Similar molecular mechanisms as described for diastemal tooth rudiments in rodents could be involved during regression of rudimental tooth primordia in the upper incisor region of the sheep.