Predicted secreted protein analysis reveals synaptogenic function of Clstn3 during WAT browning and BAT activation in mice.

Promoting white adipose tissue (WAT) browning and enhancing brown adipose tissue (BAT) activity are attractive therapeutic strategies for obesity and its metabolic complications. Targeting sympathetic innervation in WAT and BAT represents a promising therapeutic concept. However, there are few reports on extracellular microenvironment remodeling, especially changes in nerve terminal connections. Identifying the key molecules mediating the neuro-adipose synaptic junctions is a key point. In this study, we used bioinformatics methods to identify the differentially expressed predicted secreted genes (DEPSGs) during WAT browning and BAT activation. These DEPSGs largely reflect changes of cytokines, extracellular matrix remodeling, vascularization, and adipocyte-neuronal cross-talk. We then performed functional enrichment and cellular distribution specificity analyses. The upregulated and downregulated DEPDGs during WAT browning displayed a distinctive biological pattern and cellular distribution. We listed a cluster of adipocyte-enriched DEPSGs, which might participate in the cross-talk between mature adipocytes and other cells; then identified a synaptogenic adhesion molecule, Clstn3, as the top gene expressed enriched in both mature white and brown adipocytes. Using Q-PCR and immunohistochemistry, we found significantly increased Clstn3 expression level during WAT browning and BAT activation in mice subjected to cold exposure (4 °C). We further demonstrated that treatment with isoproterenol significantly increased Clstn3 and UCP1 expression in differentiated white and beige adipocytes in vitro. In conclusion, our study demonstrates that the secretion pattern was somewhat different between WAT browning and BAT activation. We reveal that Clstn3 may be a key gene mediating the neuro-adipose junction formation or remodeling in WAT browning and BAT activation process.

Comparative chemical profiling of leaf essential oils from Cinnamomum kanehirae and related species using steam distillation and solvent extraction: Implications for plant-based classification.

Cinnamomum kanehirae Hayata, belonging to Lauraceae family, is an indigenous and endangered species of considerable economic importance in Taiwan. It plays a crucial role as the host for the economically valuable saprotrophic fungus, Taiwanofungus camphorates. However, accurate species identification poses a challenge due to the similarity in morphological features and frequent natural hybridization with closely related species. Acquiring high-quality and pure leaf oils becomes imperative for precise species identification and producing superior goods. In this study, our objective was to establish methodologies for analyzing the chemical composition of leaf essential oils and subsequently apply this knowledge to differentiate among three Cinnamomum species. Gas chromatography-mass spectrometry (GC/MS) was employed to scrutinize the chemical makeup of leaf essential oils from three closely related species: C. kanehirae, C. micranthum, and C. camphora. We utilized Steam Distillation (SD) and steam distillation-solvent extraction (SDSE) methods, with the SDSE-Hexane approach chosen for optimization, enhancing extraction efficiency and ensuring essential oil purity. Through the SDSE-Hexane method, we identified seventy-four compounds distributed across three major classes: monoterpenes hydrocarbons (0.0-7.0 %), oxygenated monoterpenes (3.8-90.9 %), sesquiterpenes hydrocarbons (0.0-28.3 %), and oxygenated sesquiterpenes (1.6-88.1 %). Our findings indicated the presence of more than one chemotype in both C. kanehirae and C. camphora, whereas no specific chemotype could be discerned in C. micranthum. Furthermore, clustering based on chemotypes allowed for the differentiation of samples from the three species. Notably, we demonstrated that the chemical compositions of grafted C. kanehirae remained largely unaffected by the rootstock. Conversely, natural hybrids between C. kanehirae and C. camphora exhibited profiles more closely aligned with C. kanehirae. The optimized extraction method and the chemotype-based classification system established in this study present valuable tools for essential oil preparation, species identification, and further exploration into the genetic variation of Cinnamomum.

Microsatellite primers for the endangered beech tree, Fagus hayatae (Fagaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the endangered species Fagus hayatae (Fagaceae) to investigate the genetic diversity of the population and to investigate species delimitation within Fagus. METHODS AND RESULTS: Ten polymorphic simple sequence repeat markers were developed for F. hayatae using a magnetic bead enrichment method. The primers amplified trinucleotides, hexanucleotides, and complex repeats with six to 16 alleles per locus. The observed and expected heterozygosities across loci varied, with a range of 0.05-0.71 and 0.63-0.91, respectively. Most of the primers also amplified DNA from F. crenata, F. grandifolia, F. japonica, F. longipetiolata, F. lucida, F. orientalis, and F. sylvatica. CONCLUSIONS: These results indicate that these polymorphic loci of F. hayatae will be potentially useful for future studies of the population genetic diversity within the species. In addition, the interspecific amplification indicates that these transferable microsatellite markers will also be useful for future phylogeographic and speciation studies among close Fagus species.

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan.

PREMISE OF THE STUDY: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. METHODS AND RESULTS: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. CONCLUSIONS: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.

Structured Populations of Critically Endangered Yellow Water Lily (Nuphar shimadai Hayata, Nymphaeaceae).

Yellow water lily (Nuphar shimadai Hayata) is a critically endangered species in Taiwan. Here, we examined genetic structures of four extant populations, WP, GPa, GPb and GPn, using 39 simple sequence repeat (SSR) markers. Positive genetic correlation was observed within 50 m, beyond which no correlation was detected due to isolation by distance according to Mantel correlogram. This suggests a significant genetic structuring of the species. Besides, multilocus genotype (MLG) analysis revealed that GPa was a panmictic population and the species' putative center of origin. Genetic exchange was observed between GPa and GPb populations, which likely resulted from their geographic proximity. Nevertheless, there was a strong asymmetric migration detected from GPa to WP, but a recent genetic barrier prevented dispersal further northward (WP). Geneland estimated the best number of clusters as K = 2, where WP distinctly separated from the rest of the populations. In STRUCTURE output of K = 3, a third cluster was abundant only in WP. We suggest to consider GPn and WP as separate conservation units, being far from GPa. There is indeed a need to investigate these populations; as predicted, Ne = 1.6 to 3.0 is considered low and that may put the species at risk of extinction.

Microsatellite primers in the native perennial cycad Cycas taitungensis (Cycadaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the native perennial cycad Cycas taitungensis to evaluate the genetic variation of this endangered insular species. METHODS AND RESULTS: Using a magnetic bead enrichment method and EST data, 16 primer sets were developed and identified for the native Taiwan cycad C. taitungensis. The primers amplified dinucleotide, trinucleotide, and complex repeats with 1-9 alleles per locus. Most primers also amplified DNA from C. revoluta and C. debaoensis. CONCLUSIONS: These results indicate the utility of primers for future studies of the genetic structure of C. taitungensis. In addition, the primers are useful for further phylogeographic studies between C. taitungensis and C. revoluta, which is a closely related species.

Isolation of 16 polymorphic microsatellite markers from an endangered and endemic species, Podocarpus nakaii (Podocarpaceae).

PREMISE OF THE STUDY: Sixteen polymorphic microsatellite markers were isolated and characterized from the endangered evergreen tree Podocarpus nakaii to evaluate the population structure for conservation efforts. METHODS AND RESULTS: Based on a modified amplified fragment length polymorphism and magnetic bead enrichment method, 16 polymorphic primer sets were developed for this endangered insular species. Allele numbers ranged from five to seven, with an observed heterozygosity ranging from 0.29 to 0.88. Most primers were able to amplify DNA from the endemic P. fasciculus and the widely distributed P. macrophyllus var. macrophyllus, P. macrophyllus var. maki, and P. costalis. CONCLUSIONS: The results reported here indicate the usefulness of codominant markers for future studies of the population genetics of P. nakaii. In addition, the markers are useful for further phytogeographic and speciation studies in P. fasciculus, P. macrophyllus var. macrophyllus, and P. macrophyllus var. maki, which are closely related species.

Global dynamics and transition state theories: Comparative study of reaction rate constants for gas-phase chemical reactions.

In this review article, we present a systematic comparison of the theoretical rate constants for a range of bimolecular reactions that are calculated by using three different classes of theoretical methods: quantum dynamics (QD), quasi-classical trajectory (QCT), and transition state theory (TST) approaches. The study shows that the difference of rate constants between TST results and those of the global dynamics methods (QD and QCT) are seen to be related to a number of factors including the number of degrees-of-freedom (DOF), the density of states at transition state (TS), etc. For reactions with more DOF and higher density of states at the TS, it is found that the rate constants from TST calculations are systematically higher than those obtained from global dynamics calculations, indicating large recrossing effect for these systems. The physical insight of this phenomenon is elucidated in the present review.

Nuclear and chloroplast DNA phylogeography suggests an Early Miocene southward expansion of Lithocarpus (Fagaceae) on the Asian continent and islands.

BACKGROUND: Most genera of Fagaceae are thought to have originated in the temperate regions except for the genus Lithocarpus, the stone oaks. Lithocarpus is distributed in subtropical and tropical Asia, and its ancestral population is hypothesized to be distributed in tropical regions in Borneo and Indochina. Borneo and the nearby islands (the Greater Sunda Islands) were connected to the Malay Peninsula and Indochina prior to the Pliocene epoch and formed the former Sundaland continent. The Southeast Asian Lithocarpus, is thought to have dispersed between continental Asia and the present Sundaland. The drastic climate changes during the Pliocene and Pleistocene epochs which caused periodic sea-level changes is often used to explain the cause of its diversity. The aim of this study was to establish phylogenetic relationships by analyzing nuclear (nrDNA) and chloroplast (cpDNA) DNA in order to describe and analyze the origin, causes of diversification and historical biogeography of Lithocarpus. RESULTS: Phylogeny reconstructed through the multiple-species coalescent method with nrDNA and cpDNA revealed that the continental-Asian taxa were clustered at the basal lineages. The derived lineages of tropical Lithocarpus, with the inference of a subtropical ancestral state, imply a southward migration in the Early Miocene period with subsequent in situ diversification in the Greater Sunda Islands. The gradual decrease in temperature since the Middle Miocene period is proposed as a cause of the increase in the net diversification rate. CONCLUSIONS: The historical ancestral origin of Lithocarpus has been suggested to be mainland Asia. Southward migration in the Early Miocene period with subsequent in situ diversification could explain the current diversity of stone oaks in Southeast Asia. This study also considered the multiple origins of stone oaks currently indigenous to the subtropical islands offshore and near mainland China. Our results provide phylogenetic evidence for a subtropical origin of Asian stone oaks and reveal the process of diversification and how it fits into the timeline of major geologic and climatic events rather than local, episodic, rate-shifting events.

Characterization of 39 microsatellite markers from Nuphar shimadai (Nymphaeaceae) and cross-amplification in two related taxa.

PREMISE OF THE STUDY: Microsatellite loci were developed for Nuphar shimadai (Nymphaeaceae) to evaluate the population genetic dynamics for conservation purposes. The species is an endemic aquatic species in Taiwan that is endangered by anthropogenic activities. METHODS AND RESULTS: A magnetic bead enrichment protocol was used to identify 72 potential microsatellite loci and develop 39 microsatellite markers from N. shimadai. The number of alleles per locus ranged from one to 10 per locus, with levels of observed heterozygosity ranging from 0 to 1.0 within populations. As a result of inbreeding within isolated populations, 65% of loci significantly deviated from Hardy-Weinberg equilibrium within populations. CONCLUSIONS: These novel markers should be valuable tools to evaluate the genetic diversity within the endangered aquatic taxon N. shimadai for conservation and reintroduction purposes in Taiwan.

Development and characterization of 16 polymorphic microsatellite markers from Taiwan cow-tail fir, Keteleeria davidiana var. formosana (Pinaceae) and cross-species amplification in other Keteleeria taxa.

BACKGROUND: Keteleeria davidiana var. formosana (Pinaceae), Taiwan cow-tail fir, is an endangered species listed on the IUCN Red List of Threatened Species and only two populations remain, both on the Taiwan Island. Sixteen polymorphic microsatellite loci were developed in an endangered and endemic gymnosperm species, Keteleeria davidiana var. formosana, and were tested in an additional 6 taxa, K. davidiana var. calcarea, K. davidiana var. chienpeii, K. evelyniana, K. fortunei, K. fortunei var. cyclolepis, and K. pubescens, to evaluate the genetic variation available for conservation management and to reconstruct the phylogeographic patterns of this ancient lineage. FINDINGS: Polymorphic primer sets were developed from K. davidiana var. formosana using the modified AFLP and magnetic bead enrichment method. The number of alleles ranged from 3 to 16, with the observed heterozygosity ranging from 0.28 to 1.00. All of the loci were found to be interspecifically amplifiable. CONCLUSIONS: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct evolutionary units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.