Circadian clock genes REV-ERBα regulates the secretion of IL-1ß in deciduous tooth pulp stem cells by regulating autophagy in the process of physiological root resorption of deciduous teeth.

Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth.

The temporal protein signature analyses of developing human deciduous molar tooth germ.

The tooth serves as an exemplary model for developmental studies, encompassing epithelial-mesenchymal transition and cell differentiation. The essential factors and pathways identified in tooth development will help understand the natural development process and the malformations of mineralized tissues such as skeleton. The time-dependent proteomic changes were investigated through the proteomics of healthy human molars during embryonic stages, ranging from the cap-to-early bell stage. A comprehensive analysis revealed 713 differentially expressed proteins (DEPs) exhibiting five distinct temporal expression patterns. Through the application of weighted gene co-expression network analysis (WGCNA), 24 potential driver proteins of tooth development were screened, including CHID1, RAP1GDS1, HAPLN3, AKAP12, WLS, GSS, DDAH1, CLSTN1, AFM, RBP1, AGO1, SET, HMGB2, HMGB1, ANP32A, SPON1, FREM1, C8B, PRPS2, FCHO2, PPP1R12A, GPALPP1, U2AF2, and RCC2. Then, the proteomics and transcriptomics expression patterns of these proteins were further compared, complemented by single-cell RNA-sequencing (scRNA-seq). In summary, this study not only offers a wealth of information regarding the molecular intricacies of human embryonic epithelial and mesenchymal cell differentiation but also serves as an invaluable resource for future mechanistic inquiries into tooth development.

Weak evidence of provenance effects in spring phenology across Europe and North America.

Forecasting the biological impacts of climate change requires understanding how species respond to warmer temperatures through interannual flexible variation vs through adaptation to local conditions. Yet, we often lack this information entirely or find conflicting evidence across studies, which is the case for spring phenology. We synthesized common garden studies across Europe and North America that reported spring event dates for a mix of angiosperm and gymnosperm tree species in the northern hemisphere, capturing data from 384 North American and 101 European provenances (i.e. populations) with observations from 1962 to 2019, alongside autumn event data when provided. Across continents, we found no evidence of provenance effects in spring phenology, but strong clines with latitude and mean annual temperature in autumn. These effects, however, appeared to diverge by continent and species type (gymnosperm vs angiosperm), with particularly pronounced clines in North America in autumn events. Our results suggest flexible, likely plastic responses, in spring phenology with warming, and potential limits - at least in the short term - due to provenance effects for autumn phenology. They also highlight that, after over 250 yr of common garden studies on tree phenology, we still lack a holistic predictive model of clines across species and phenological events.

Ecological drivers of flower-leaf sequences: aridity and proxies for pollinator attraction select for flowering-first in the American plums.

Across temperate forests, many tree species produce flowers before their leaves emerge. This flower-leaf phenological sequence, known as hysteranthy, is generally described as an adaptation for wind pollination. However, this explanation does not address why hysteranthy is also common in biotically pollinated taxa. We quantified flower-leaf sequence variation in the American plums (Prunus, subg. Prunus sect. Prunocerasus), a clade of insect-pollinated trees, using herbaria specimens and Bayesian hierarchical modeling. We tested two common, but rarely interrogated hypotheses - that hysteranthy confers aridity tolerance and/or pollinator visibility - by modeling the associations between hysteranthy and related traits. To understand how these phenology-trait associations were sensitive to taxonomic scale and flower-leaf sequence classification, we then extended these analyses to all Prunus species in North America. Our findings across two taxonomic levels support the hypotheses that hysteranthy may help temporally partition hydraulic demand to reduce water stress and increase pollinator visibility - thereby reducing selective pressure on inflorescence size. Our results provide foundational insights into the evolution of flower-leaf sequences in the genus Prunus, with implications for understanding these patterns in biotically pollinated plants in general. Our approach suggests a path to advance these hypotheses to other clades, but teasing out drivers fully will require new experiments.

Hydraulic plasticity and water use regulation act to maintain the hydraulic safety margins of Mediterranean trees in rainfall exclusion experiments.

Hydraulic failure due to xylem embolism has been identified as one of the main mechanisms involved in drought-induced forest decline. Trees vulnerability to hydraulic failure depends on their hydraulic safety margin (HSM). While it has been shown that HSM globally converges between tree species and biomes, there is still limited knowledge regarding how HSM can adjust locally to varying drought conditions within species. In this study, we relied on three long-term partial rainfall exclusion experiments to investigate the plasticity of hydraulic traits and HSM for three Mediterranean tree species (Quercus ilex L., Quercus pubescens Willd., and Pinus halepensis Mill.). For all species, a homeostasis of HSM in response to rainfall reduction was found, achieved through different mechanisms. For Q. ilex, the convergence in HSM is attributed to the adjustment of both the turgor loss point (Ψtlp) and the water potential at which 50% of xylem conductivity is lost due to embolism (P50). In contrast, the maintenance of HSM for P. halepensis and Q. pubescens is related to its isohydric behavior for the first and leaf area adjustment for the latter. It remains to be seen whether this HSM homeostasis can be generalized and if it will be sufficient to withstand extreme droughts expected in the Mediterranean region.

New craniodental fossils of Paranthropus robustus from Kromdraai, South Africa (2014-2017 excavations).

Since the initial discovery of Paranthropus robustus at the site of Kromdraai in 1938, the hypodigm of this species has been expanded by subsequent work at the localities of Swartkrans and Drimolen, with a few fossils also known from Cooper's D, Gondolin and Sterkfontein Member 5. Beginning in 2014, systematic excavations at Kromdraai uncovered a large and previously unknown fossiliferous area, shedding light on Units O and P in the earliest part of the site's stratigraphic sequence. The aim of this paper is to provide detailed descriptions and illustrations of 30 P. robustus craniodental specimens recovered between 2014 and 2017 within the Unit P deposits at Kromdraai. This new sample predates all prior conspecific specimens found at this site (including the holotype of P. robustus from Kromdraai, TM 1517). Its basic dental morphology dimensions and cranial features are compared in a preliminary analysis with other P. robustus samples. The P. robustus sample from Kromdraai Unit P documents previously unknown portions of the P. robustus juvenile cranium. The new dental and cranial remains aid in the exploration of potential morphological distinctions between site-specific P. robustus samples and are compared favorably in size and morphology with the small P. robustus specimens from Drimolen (e.g., DNH 7). These findings do not support the hypothesis that the specimens from Drimolen belong to a different taxonomic group. Instead, they reinforce the presence of a significant degree of sexual dimorphism within P. robustus. The Kromdraai Unit P specimens also contribute to the biodemographic profile of P. robustus. The notable prevalence of infants (i.e., juvenile individuals before the emergence of their first permanent molars) mirrors the natural mortality profiles observed in wild chimpanzees. This suggests a closer resemblance in the processes of accumulation in Kromdraai Unit P and Drimolen than at Swartkrans.

Exosomes miR-92a-3p from human exfoliated deciduous teeth inhibits periodontitis progression via the KLF4/PI3K/AKT pathway.

BACKGROUND: Periodontitis is a chronic inflammatory disease mediated by dysbiosis of the oral microflora, resulting in the destruction of periodontal tissue. Increasing evidence suggested that mesenchymal stem cell (MSCs) and exosomes derived from MSCs play a critical role in periodontal tissue regeneration. However, whether stem cells from exfoliated deciduous teeth (SHED)-secreted exosomes can improve the therapeutic potential of periodontitis is largely unknown. OBJECTIVE: Here, we aim to evaluate the effect of SHED-exosomes on inflammation, apoptosis and osteogenic differentiation in periodontitis. METHODS: The periodontitis cell model was constructed by stimulating periodontal ligament stem cells (PDLSCs) with lipopolysaccharide (LPS), and the periodontitis rats were established by ligation. RESULTS: First, we isolated exosomes from the SHED, and we figured out that exosomes secreted by SHED were enriched in miR-92a-3p and the exosomes enhanced proliferation and osteogenic differentiation and reduced apoptosis and inflammatory responses in PDLSCs. In addition, we found that SHED-exosomes alleviated inflammatory effect and elevated the expression of osteogenic-related genes in periodontitis rat model. Moreover, miR-92a-3p targeted downstream Krüppel-Like Transcription Factor 4 (KLF4) and regulated the PI3K/AKT pathway. Finally, our data indicated that upregulation of KLF4 or activation of PI3K/AKT by 740Y-P counteracted the inhibitory effect of SHED-exosomes on periodontitis progression. CONCLUSION: Taken together, our finding revealed that exosomal miR-92a-3p derived from SHED contributed to the alleviation of periodontitis development and progression through inactivating the KLF4/PI3K/AKT signaling pathway, which may provide a potential target for the treatment of periodontitis.

Deciphering the genetic mechanisms of chilling requirement for bud endodormancy release in deciduous fruit trees.

Bud endodormancy in deciduous fruit trees is an adaptive trait evolved by selection for the capacity to survive unfavorable environmental conditions. Deciduous trees require a certain amount of winter chill named chilling requirement (CR) to promote bud endodormancy release. In recent decades, global warming has endangered the chill accumulation in deciduous fruit trees. Developing low-CR cultivars is a practical way to neutralize the effect of climate changes on the cultivation and distribution of deciduous fruit trees. In this review, we focus on the effect of chilling accumulation on bud endodormancy release and the genetic mechanisms underlying the chilling requirement in deciduous fruit trees. Additionally, we put forth a regulatory model for bud endodormancy and provide prospective directions for future research in deciduous fruit trees.

Deciduous teeth from the New Hampshire birth cohort study: Early life environmental and dietary predictors of dentin elements.

BACKGROUND: Sparse research exists on predictors of element concentrations measured in deciduous teeth. OBJECTIVE: To estimate associations between maternal/child characteristics, elements measured in home tap water during pregnancy and element concentrations in the dentin of shed deciduous teeth. METHODS: Our analysis included 152 pregnant person-infant dyads followed from the second trimester through the end of the first postnatal year from the New Hampshire Birth Cohort Study. During pregnancy and early infancy, we collected dietary and sociodemographic information via surveys, measured elements in home tap water, and later collected naturally exfoliated teeth from child participants. We measured longitudinal deposition of elements in dentin using LA-ICP-MS. Multivariable linear mixed models were used to estimate associations between predictors and dentin element concentrations. RESULTS: We measured 12 elements in dentin including those previously reported (Ba, Mn, Pb, Sr, Zn) and less frequently reported (Al, As, Cd, Cu, Hg, Li, and W). A doubling of Pb or Sr concentrations in water was associated with higher dentin Pb or Sr respectively in prenatally formed [9% (95%CI: 3%, 15%); 3% (1%, 6%)] and postnatally formed [10% (2%, 19%); 6% (2%, 10%)] dentin. Formula feeding from birth to 6 weeks or 6 weeks to 4 months was associated with higher element concentrations in postnatal dentin within the given time period as compared to exclusive human milk feeding: Sr: 6 weeks: 61% (36%, 90%) and 4 months: 85% (54%, 121%); Ba: 6 weeks: 35% (3.3%, 77%) and 4 months: 42% (10%, 83%); and Li: 6 weeks: 61% (33%, 95%) and 4 months: 58% (31%, 90%). SIGNIFICANCE: These findings offer insights into predictors of dentin elements and potential confounders in exposure-health outcome relationships during critical developmental periods.

Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration.

BACKGROUND: Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration. RESULTS: We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). CONCLUSIONS: The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics.

BACKGROUND: Human mesenchymal stem cells have attracted interest in regenerative medicine and are being tested in many clinical trials. In vitro expansion is necessary to provide clinical-grade quantities of mesenchymal stem cells; however, it has been reported to cause replicative senescence and undefined dysfunction in mesenchymal stem cells. Quality control assessments of in vitro expansion have rarely been addressed in ongoing trials. Young small extracellular vesicles from the remnant pulp of human exfoliated deciduous teeth stem cells have demonstrated therapeutic potential for diverse diseases. However, it is still unclear whether young small extracellular vesicles can reverse senescence-related declines. RESULTS: We demonstrated that mitochondrial structural disruption precedes cellular dysfunction during bone marrow-derived mesenchymal stem cell replication, indicating mitochondrial parameters as quality assessment indicators of mesenchymal stem cells. Dynamin-related protein 1-mediated mitochondrial dynamism is an upstream regulator of replicative senescence-induced dysfunction in bone marrow-derived mesenchymal stem cells. We observed that the application of young small extracellular vesicles could rescue the pluripotency dissolution, immunoregulatory capacities, and therapeutic effects of replicative senescent bone marrow-derived mesenchymal stem cells. Mechanistically, young small extracellular vesicles could promote Dynamin-related protein 1 translocation from the cytoplasm to the mitochondria and remodel mitochondrial disruption during replication history. CONCLUSIONS: Our findings show that Dynamin-related protein 1-mediated mitochondrial disruption is associated with the replication history of bone marrow-derived mesenchymal stem cells. Young small extracellular vesicles from human exfoliated deciduous teeth stem cells alleviate replicative senescence by promoting Dynamin-related protein 1 translocation onto the mitochondria, providing evidence for a potential rejuvenation strategy.

Biomimetic pulp scaffolds prepared from extracellular matrix derived from stem cells from human exfoliated deciduous teeth promote pulp-dentine complex regeneration.

AIM: To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED-ECM-PS) in promoting pulp-dentine complex regeneration. METHODOLOGY: SHED-ECM-PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED-ECM-PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED-ECM-PS on regeneration of the pulp-dentine complex in vivo. Extraction medium for SHED-ECM-PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed in vitro. Cell counting kit-8 and Ki-67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two-tailed Student's t-test. p < .05 was considered statistically significant. RESULTS: SHED-ECM-PS was successfully constructed, exhibiting a striped dental pulp-like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED-ECM-PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED-ECM-PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast-like cells was observed along the inner wall of the root canal. SHED-ECM-PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group in vitro. CONCLUSION: SHED-ECM-PS exhibited no cytotoxicity and promoted pulp-dentine complex regeneration in vivo as well as cell migration and odontogenic differentiation of BMMSCs in vitro. These findings provide evidence that SHED-ECM-PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs.

Characterizing spring phenology in a temperate deciduous urban woodland fragment: trees and shrubs.

Phenological research in temperate-deciduous forests typically focuses on upper canopy trees, due to their overwhelming influence on ecosystem productivity and function. However, considering that shrubs leaf out earlier and remain green longer than trees, they play a pivotal role in ecosystem productivity, particularly at growing season extremes. Furthermore, an extended growing season of non-native shrubs provides a competitive advantage over natives. Here, we report spring phenology, budburst, leaf-out, and full-leaf unfolded (2017-2021) of a range of co-occurring species of tree (ash, American basswood, red oak, white oak, and boxelder) and shrub (native species: chokecherry, pagoda dogwood, nannyberry, American wild currant and Eastern wahoo, and non-native species: buckthorn, honeysuckle, European privet, and European highbush cranberry) in an urban woodland fragment in Wisconsin, USA, to determine how phenology differed between plant groups. Our findings show that all three spring phenophases of shrubs were 3 weeks earlier (p < 0.05) than trees. However, differences between shrubs groups were only significant for the later phenophase; full-leaf unfolded, which was 6 days earlier (p < 0.05) for native shrubs. The duration of the spring phenological season was 2 weeks longer (p < 0.05) for shrubs than trees. These preliminary findings demonstrate that native shrubs, at this site, start full-leaf development earlier than non-native species suggesting that species composition must be considered when generalizing whether phenologies differ between vegetation groups. A longer time series would be necessary to determine future implications on ecosystem phenology and productivity and how this might impact forests in the future, in terms of species composition, carbon sequestration, and overall ecosystem dynamics.

Variation in the timing and duration of autumn leaf phenology among temperate deciduous trees, native shrubs and non-native shrubs.

The timing and duration of autumn leaf phenology marks important transitions in temperate deciduous forests, such as, start of senescence, declining productivity and changing nutrient cycling. Phenological research on temperate deciduous forests typically focuses on upper canopy trees, overlooking the contribution of other plant functional groups like shrubs. Yet shrubs tend to remain green longer than trees, while non-native shrubs, in particular, tend to exhibit an extended growing season that confers a competitive advantage over native shrubs. We monitored leaf senescence and leaf fall (2017-2020) of trees and shrubs (native and non-native) in an urban woodland fragment in Wisconsin, USA. Our findings revealed that, the start of leaf senescence did not differ significantly between vegetation groups, but leaf fall started (DOY 273) two weeks later in shrubs. Non-native shrubs exhibited a considerably delayed start (DOY 262) and end of leaf senescence (DOY 300), with leaf-fall ending (DOY 315) nearly four weeks later than native shrubs and trees. Overall, the duration of the autumn phenological season was longer for non-native shrubs than either native shrubs or trees. Comparison of the timing of spring phenophases with the start and end of leaf senescence revealed that when spring phenology in trees starts later in the season senescence also starts later and ends earlier. The opposite pattern was observed in native shrubs. In conclusion, understanding the contributions of plant functional groups to overall forest phenology requires future investigation to ensure accurate predictions of future ecosystem productivity and help address discrepancies with remote sensing phenometrics.

Arbuscular mycorrhizal fungal diversity and potential association networks among African tropical forest trees.

Tropical forests represent one of the most diverse and productive ecosystems on Earth. High productivity is sustained by efficient and rapid cycling of nutrients, which is in large part made possible by symbiotic associations between plants and mycorrhizal fungi. In these associations, an individual plant typically associates simultaneously with multiple fungi and the fungi associate with multiple plants, creating complex networks among fungi and plants. However, there are few studies that have investigated mycorrhizal fungal composition and diversity in tropical forest trees, particularly in Africa, or that assessed the structure of the network of associations among fungi and trees. In this study, we collected root and soil samples from Ise Forest Reserve (Southwest Nigeria) and used a metabarcoding approach to identify the dominant arbuscular mycorrhizal (AM) fungal taxa in the soil and associating with ten co-occurring tree species to assess variation in AM communities. Network analysis was used to elucidate the architecture of the network of associations between fungi and tree species. A total of 194 Operational Taxonomic Units (OTUs) belonging to six AM fungal families were identified, with 68% of all OTUs belonging to Glomeraceae. While AM fungal diversity did not differ among tree species, AM fungal community composition did. Network analyses showed that the network of associations was not significantly nested and showed a relatively low level of specialization (H2 = 0.43) and modularity (M = 0.44). We conclude that, although there were some differences in AM fungal community composition, the studied tree species associate with a large number of AM fungi. Similarly, most AM fungi had great host breadth and were detected in most tree species, thereby potentially working as interaction network hubs.

Shear bond strength of a composite resin restoration in primary teeth following cavity preparation using laser- an in-vitro study.

To evaluate and compare the shear bond strength of composite resin restorations in primary teeth, following cavity preparation with both traditional dental burs and laser irradiation. One hundred primary molars extracted from the children visiting our department were collected and randomly divided into five groups (A-E) with 20 teeth in each group. In groups A, B, C, D, and E the teeth samples were etched with phosphoric acid, Er; YAG laser followed by acid etching, Er, Cr: YSGG laser followed by acid etching, Er; YAG laser etching only and Er, Cr: YSGG laser etching only, respectively. Following, all the samples were restored with composite resin and subjected to 500 cycles of thermocycling. The shear bond strength of the resin composite was analyzed. The type of fractures was also noted. Data obtained were subjected to statistical analysis. The mean value of shear bond strength of Group A, B, C, D, and E was 17.562 ± 0.810, 15.928 ± 0.415, 14.964 ± 0.566, 11.833 ± 0.533 and 11.187 ± 0.517, respectively. Adhesive failure was most commonly seen in all the groups. The phosphoric acid etching remains a highly effective technique for pre-treating dentin in composite resin restorations. The shear strength of composite resin to the dentin of laser-prepared cavity in primary teeth can be improved by the addition of acid etching.

Leaf Spot Disease of Michelia alba caused by Alternaria alternata in China.

The perennial deciduous tree Michelia alba is a widely cultivated street plant in China. In June 2021 and March 2022, M. alba trees with leaf spots were found in the green belt of the approximately 200,000 m2 community (32.62°N, 116.98°E) of Tianjia'an District, Huainan, Anhui, China, where approximately half of the M. alba trees had brown leaf spots surrounded with irregular yellow halos ranging from 2 to 6 mm in diameter (Fig S1A). The leaves of M. alba trees with multiple lesions became blighted. To isolate the potential pathogens causing leaf spot symptoms in M. alba trees, twenty fragments (2 cm2) were excised from the margin of the necrosis on symptomatic leaves, immersed in 1% sodium hypochlorite for 45 s, and then washed three times in sterile water. The fragments were plated and incubated on potato dextrose agar (PDA) at 25 °C and 15 dark green fungal colonies were obtained 5 days later. Single-spore isolates of the fungal colonies plated on potato carrot agar (PCA, Simmons 2007) produced gray, floccose colonies, which reached 71 mm after 7 days at 25 °C were obtained 5 days later (Fig S1C). Optical microscopy analysis showed that single-spore isolates formed sparsely branched chains with pale brown conidiophores on PCA after incubation at 25 °C in darkness for 7 days. The conidia were ellipsoidal, inverted rod, or ovoid, light brown, and 10.0 to 52.5 × 4.5 to 22.7 µm, with zero to four longitudinal or oblique and zero to eight transverse septa (n = 50). Partial conidia are 2.5 to 27.5 × 0.6 to 3.7 µm with cylindrical light brown beaks (n = 50) (Fig S1D, E). The cultural and morphological characteristics of the isolated fungi were consistent with the description of Alternaria alternata (Woudenberg et al. 2015). To further characterize the isolated fungi, the genomic DNA of three representative strains (BYL-1, BYL-2 and BYL-3) were extracted from their mycelia, respectively. ITS region and housekeeping genes GPD, and TEF, were amplified and sequenced using ITS4/ITS5 (White et al. 1990), Gpd1/Gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), primer pairs, respectively. BLAST analysis showed that the isolates BYL-1 (GenBank accession nos. OP325693, OP405008, and OP405009), BYL-2 (GenBank accession nos. PP057859, PP138442, and PP138444), and BYL-3 (GenBank accession nos. PP057860, PP138443, and PP138445) shared 99 to 100% identity with Alternaria alternata (GenBank accession nos. AF347032.1, AY278809.1, KC584693.1), which suggested that all the three isolates belong to A. alternata. The identifications were further confirmed by phylogenetic analysis based on combined DNA sequences data of ITS, GPD, and TEF. As showed in Fig S2, the strains of BYL-1 , BYL-2 and BYL-3 formed a robust clade with A. alternata CBS918.96. Taken together, the morphology and molecular assays suggest that strain BYL-1 is A. alternata. To test pathogenicity, the isolate BYL-1 was cultured on PCA for 7 days to prepare conidial suspensions, and the spore concentration was adjust to a final concentration of 105 spores/ml. The leaves of 3-5-leaf stage of six 5-years-old natural planting M. alba plants were sprayed with conidial suspensions and sterile distilled water, respectively. The petiole of each inoculated leaves of M. alba were secured with sterile wet cotton, and covered with plastic bags to prevent moisture evaporation after incubation. After a 3- to 5- day of inoculation, necrotic lesions appeared on the leaves inoculated with conidial suspensions, whereas no necrotic lesion was observed in the control leaves inoculated with sterile distilled water (Fig S1B). To fulfill the Koch,s postulates, fungi were re-isolated from the margin of necrotic lesions and identified as A. alternata by DNA sequencing the ITS gene. To our knowledge, this is the first report of A. alternata causing leaf spot on M. alba. Because the disease could cause damage to the foliage influencing the greening and ornamental effects of these trees, control measures may need to be implemented during daily management.

An examination of Homo naledi early juveniles recovered from the Rising Star cave system, South Africa.

BACKGROUND: Six Homo naledi early juveniles were recovered from U.W. 101 (Dinaledi Chamber), U.W. 102 (Lesedi Chamber), and U.W. 110 in the Rising Star cave system. AIM: This paper develops the information for the H. naledi early juvenile life stage, as defined by a combination of deciduous and permanent dentition, and the eruption of the first permanent molar. SUBJECTS AND METHODS: The growing number of young individuals recovered from the Rising Star cave system allows us to gain a better understanding of their variation, or lack thereof, and provides a basis to estimate broad ranges for age at death of the individuals. The individuals are identified and described through craniodental remains and spatial associations. RESULTS AND CONCLUSION: Our results show that the teeth are remarkably consistent across the localities in their metric and non-metric traits, and our analyses refine previous estimations on dental eruptions with the first permanent molar erupting first in the sequence among permanent teeth.

Agenesis of the maxillary permanent lateral incisors with the deciduous retained: Conservative and biomimetic approach using the BAIR technique.

OBJECTIVE: Agenesis of the maxillary permanent lateral incisors is a condition that requires treatment aimed at improving the esthetics, even at an early age. However, traditional therapeutic protocols are long, invasive and have limitations and contraindications imposed by the age of the patient. CLINICAL CONSIDERATIONS: Recent developments in restorative dentistry have provided a new approach to this clinical situation, in particular when the deciduous laterals are retained. We report two cases regarding the management of missing lateral incisors using Biologically Active Intrasulcular Restoration (BAIR) technique. The BAIR technique allows us to transform the shape of the deciduous lateral incisor into the permanent, acting both on the dental morphology and proportions, and on the appearance of the soft tissues and the gingival parables. CONCLUSIONS: The BAIR technique is a valid approach to cases of agenesis of the maxillary permanent lateral incisors, when the deciduous are retained. It does not require any preparation of the dental tissues, is reversible and minimally invasive. It is applicable to patients of all ages, and results are obtained in a single appointment. CLINICAL SIGNIFICANCE: The BAIR technique allows a biomimetic conservative approach for the rehabilitation of congenitally missing permanent lateral incisors, when the deciduous are retained. It is a non-invasive protocol and effective in successfully restoring esthetics.

The Exosomes of Stem Cells from Human Exfoliated Deciduous Teeth Suppress Inflammation in Osteoarthritis.

Hyaluronic acid injection is commonly used clinically to slow down the development of osteoarthritis (OA). A newly developed therapeutic method is to implant chondrocytes/stem cells to regenerate cartilage in the body. The curative effect of stem cell therapy has been proven to come from the paracrine of stem cells. In this study, exosomes secreted by stem cells from human exfoliated deciduous teeth (SHED) and hyaluronic acid were used individually to evaluate the therapeutic effect in slowing down OA. SHED was cultured in a serum-free medium for three days, and the supernatant was collected and then centrifuged with a speed difference to obtain exosomes containing CD9 and CD63 markers, with an average particle size of 154.1 nm. SW1353 cells were stimulated with IL-1ß to produce the inflammatory characteristics of OA and then treated with 40 µg/mL exosomes and hyaluronic acid individually. The results showed that the exosomes successfully inhibited the pro-inflammatory factors, including TNF-α, IL-6, iNOS, NO, COX-2 and PGE2, induced by IL-1ß and the degrading enzyme of the extrachondral matrix (MMP-13). Collagen II and ACAN, the main components of the extrachondral matrix, were also increased by 1.76-fold and 2.98-fold, respectively, after treatment, which were similar to that of the normal joints. The effect can be attributed to the partial mediation of SHED exosomes to the NF-κB pathway, and the ability of exosomes to inhibit OA is found not inferior to that of hyaluronic acid.