Speciation patterns of related species under the hybrid zone: A case study of three sclerophyllous oaks in the east Himalaya-Hengduan Mountains.

Speciation is often accompanied by frequent gene exchanges, which have been gradually recognized as a common phenomenon in nature. Although gene flow may influence different reproductive isolations, the specific mechanism of the process still requires more experimental evidence, especially in hybrid populations that have no significant differentiation and isolation. To address this challenge, this study aims to elucidate the underlying mechanisms of sympatry and parapatry in related species. Three sclerophyllous oaks (Quercus spinosa, Quercus aquifolioides and Quercus rehderiana), which are mainly distributed in the sympatry/parapatry of the East Himalaya-Hengduan Mountains and adjacent regions, were used to explore the population dynamics and evolutionary history. Based on 12,420 genome-wide single nucleotide polymorphism datasets, gene flow detection showed that the three species did not present any obvious genetic barriers. Evolutionary analysis indicated that the three species diverged during the Tertiary Period, and no migration events occurred in the early stages of species divergence. Combined with the data of 19 ecological factors, geological movements and climatic turbulence caused the rapid radiated differentiation of the three species during the Neocene, and similar selective pressures resulted in the same evolutionary pattern based on demographic history analysis. In addition, the predicted niche occupancy profiles and Generalized Dissimilarity Modelling revealed that the three species occupied distinct niches and had significant differences in ecological adaptation, which may explain the specific morphological characteristics of the different species. Therefore, we believe that the populations of the three related species underwent adaptive evolution in different habitats during the early stages of divergence. This study provides new experimental evidence of the formation patterns of parallel speciation.

Lycopene abolishes typical polyhalogenated carbazoles (PHCZs)-induced hepatic injury in yellow catfish (Pelteobagrus fulvidraco): Involvement of ROS/PI3K-AKT/NF-κB signaling.

Aquatic ecosystems are being more contaminated with polyhalogenated carbazoles (PHCZs), which raising concerns about their impact on aquatic organisms. Lycopene (LYC) exhibits several beneficial properties for fish via enhance antioxidant defenses and improve immunity. In this study, we attempted to investigate the hepatotoxic effects of typical PHCZs 3, 6-dichlorocarbazole (3,6-DCCZ) and the protective mechanisms of LYC. In this study, we found that yellow catfish (Pelteobagrus fulvidraco) exposure to 3,6-DCCZ (1.2 mg/L) resulted in hepatic inflammatory infiltration and disordered hepatocyte arrangement. Besides, we observed that 3,6-DCCZ exposure resulted in hepatic reactive oxygen species (ROS) overproduction and excessive autophagosome accumulation, accompanied with inhibition of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway. Subsequently, we confirmed that 3,6-DCCZ exposure triggered hepatic uncontrolled inflammatory response via activation of nuclear factor-κB (NF-κB) pathway, along with decreased plasma complement C3 (C3) and complement C4 (C4) levels. Meanwhile, yellow catfish exposed to 3,6-DCCZ exhibit an increased hepatic apoptosis phenomenon, as evidenced by the elevated number of positive TUNEL cells and upregulated expression of caspase3 and cytochrome C (CytC). In contrast, LYC treatment could alleviate the 3,6-DCCZ-induced pathological changes, hepatic ROS accumulation, autophagy, inflammatory response and apoptosis. To sum up, this study provided the demonstration that LYC exerts hepatoprotective effects to alleviate 3,6-DCCZ-induced liver damage by inihibiting ROS/PI3K-AKT/NF-κB signaling in yellow catfish.

Tandem mass tag (TMT) labeling-based quantitative proteomic analysis reveals the cellular protein characteristics of 16HBE cells infected with coxsackievirus A10 and the potential effect of HMGB1 on viral replication.

Coxsackievirus A10 (CV-A10) is recognized as one of the most important pathogens associated with hand, foot, and mouth disease (HFMD) in young children under 5 years of age worldwide, and it can lead to fatal neurological complications. However, available commercial vaccines fail to protect against CV-A10. Therefore, there is an urgent need to study new protein targets of CV-A10 and develop novel vaccine-based therapeutic strategies. Advances in proteomics in recent years have enabled a comprehensive understanding of host pathogen interactions. Here, to study CV-A10-host interactions, a global quantitative proteomic analysis was conducted to investigate the molecular characteristics of host cell proteins and identify key host proteins involved in CV-A10 infection. Using tandem mass tagging (TMT)-based mass spectrometry, a total of 6615 host proteins were quantified, with 293 proteins being differentially regulated. To ensure the validity and reliability of the proteomics data, three randomly selected proteins were verified by Western blot analysis, and the results were consistent with the TMT results. Further functional analysis showed that the upregulated and downregulated proteins were associated with diverse biological activities and signaling pathways, such as metabolic processes, biosynthetic processes, the AMPK signaling pathway, the neurotrophin signaling pathway, the MAPK signaling pathway, and the GABAergic synaptic signaling. Moreover, subsequent bioinformatics analysis demonstrated that these differentially expressed proteins contained distinct domains, were localized in different subcellular components, and generated a complex network. Finally, high-mobility group box 1 (HMGB1) might be a key host factor involved in CV-A10 replication. In summary, our findings provide comprehensive insights into the proteomic profile during CV-A10 infection, deepen our understanding of the relationship between CV-A10 and host cells, and establish a proteomic signature for this viral infection. Moreover, the observed effect of HMGB1 on CV-A10 replication suggests that it might be a potential therapeutic target treatment of CV-A10 infection.

Ferroptosis contribute to neonicotinoid imidacloprid-evoked pyroptosis by activating the HMGB1-RAGE/TLR4-NF-κB signaling pathway.

Imidacloprid (IMI) is among the common neonicotinoid insecticides used in agriculture worldwide, posing a potential toxic threat to non-target animals and humans. Numerous studies have shown that ferroptosis is involved in the pathophysiological progression of renal diseases. However, it remains unclear whether ferroptosis is involved in IMI-induced nephrotoxicity. In the present study, we investigated the potential pathogenic role of ferroptosis in IMI-induced kidney damage in vivo. Transmission electron microscopy (TEM) showed that the mitochondrial crest of kidney cells significantly decreased following IMI exposure. Moreover, IMI exposure triggered ferroptosis and lipid peroxidation in the kidney. We confirmed that nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant capability was negatively correlated with the ferroptosis induced by IMI exposure. Importantly, we verified that NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3)-driven inflammation occurred in the kidneys following IMI exposure, but pretreatment with the ferroptosis inhibitor ferrostatin (Fer-1) blocked this phenomenon. Additionally, IMI exposure induced F4/80+ macrophages to accumulated in the proximal tubules of the kidneys, and also increased the protein expression of high-mobility group box 1 (HMGB1), receptor for advanced glycation end products (RAGE), receptor for advanced glycation end products (TLR4), and nuclear factor kappa-B (NF-κB). In contrast, inhibition of ferroptosis by Fer-1 blocked IMI-induced NLRP3 inflammasome activation, F4/80 positive macrophages, and the HMGB1-RAGE/TLR4 signaling pathway. To the best of our knowledge, this is the first study to reveal that IMI stress can induce Nrf2 inactivation, thereby triggering ferroptosis, causing an initial wave of death, and activating HMGB1-RAGE/TLR4 signaling, which promotes pyroptosis that perpetuates kidney dysfunction.

Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease that has become the third leading cause of death worldwide. Cycloastragenol (CAG), which is the genuine sapogenin of the main active triterpene saponins in Astragali radix, is a bioavailable pre-clinical candidate for chronic obstructive pulmonary disease (COPD), and it was investigated in our previous study. In order to progress medical research, it was first efficiently produced on a 2.5-kg scale via Smith degradation from astragaloside IV (AS-IV). Simultaneously, since the impurity profiling of a drug is critical for performing CMC documentation in pre-clinical development, a study on impurities was carried out. As these structures do not contain chromophores and possess weak UV absorption characteristics, HPLC-CAD and UPLC-LTQ-Orbitrap-MS were employed to carry out the quality control of the impurities. Then, column chromatography (CC), preparative thin-layer chromatography (PTLC), and crystallization led to the identification of 15 impurities from CAG API. Among these impurities, compounds 1, 4, 9, 10, 14, and 15 were elucidated via spectroscopic analysis, and 2-3, 5-8, and 11-13 were putatively identified. Interestingly, the new compounds 9 and 14 were rare 10, 19-secocycloartane triterpenoids that displayed certain anti-inflammatory activities against LPS-induced lymphocyte cells and CSE-induced MLE-12 cells. Additionally, a plausible structural transformation pathway of the degradation compounds from CAG or AS IV was proposed. The information obtained will provide a material basis to carry out the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided regarding the compounds with weak UV absorption characteristics.

Phylogenetic relationships in Chinese oaks (Fagaceae, Quercus): Evidence from plastid genome using low-coverage whole genome sequencing.

China is a second center of oak diversity but with less intensively systematic studies. Here, with 49 species representing all four sections in China, we firstly gave insight into the comprehensive phylogenetic relationships of Chinese oaks based on 54 complete plastid genomes. Our results recovered a robust phylogenetic framework and provided strong support for most nodes. The phylogenetic tree supported Quercus section Ilex as not monophyletic, in which Quercus section Cyclobalanopsis and Quercus section Cerris were nested. Most likely, incomplete lineage sorting and/or introgression among ancestral lineages in these three sections resulted in this complex pattern. The current distribution, diversification and molecular differentiation of Q. sect. Ilex in China are likely consequences of local adaptation to the geographic and paleoclimatic changes, which were driven by the uplift of Tibetan Plateau, the Hengduan Mountains and the Himalayas.

Transcriptome and metabolome analysis of stress tolerance to aluminium in Vitis quinquangularis.

MAIN CONCLUSION: Transcriptional and metabolic regulation of aluminium tolerance of Chinese wild Vitis quinquangularis after Al treatment for 12 h: genes and pathways related to stress resistance are activated to cope with Al stress. The phytotoxicity of aluminium (Al) has become a major issue in inhibiting plant growth in acidic soils. Chinese wild Vitis species have excellent stress resistance. In this study, to explore the mechanism underlying Al tolerance in Chinese wild Vitis quinquangularis, we conducted a transcriptome analysis to understand the changes in gene expression and pathways in V. quinquangularis leaves after Al treatment for 12 h (Al_12 h). Compared with the control (CK) treatment, 2266 upregulated differentially expressed genes (DEGs) and 2943 downregulated DEGs were identified after Al treatment. We analysed the top 60 upregulated DEGs and found that these genes were related mostly to cell wall organization or biogenesis, transition metal ion binding, etc. Another analysis of all the upregulated DEGs showed that genes related to the ABC transport pathway, salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) hormone signalling pathway were expressed. Transcriptome and metabolome analysis revealed that genes and metabolites (phenylalanine, cinnamate and quercetin) related to the phenylalanine metabolic pathway were expressed. In summary, the results provide a new contribution to a better understanding of the metabolic changes that occur in grapes after Al stress as well as to research on improving the resistance of grape cultivars.

Paenibacillus turpanensis sp. nov., isolated from a salt lake of Turpan city in Xinjiang province, north-west China.

Strain YIM B00363T, a Gram-positive, aerobic, non-motile, rod-shaped, spore-forming bacterium, was isolated from saline soil samples collected from a salt lake in Xinjiang province, north-west China, and was characterized using a polyphasic approach. The optimum growth temperature was 37 °C and the optimum pH was 7.5-8.0. The major menaquinone was MK-7; anteiso-C15:0 (53.52%), iso-C15:0 (15.04%) and C16:0 (12.76%) were the predominant cellular fatty acids. The diagnostic diamino acid of the cell wall peptidoglycan was meso-diaminopimelic acid. The phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, unidentified glycolipids and unknown lipids. The DNA G + C content of the type strain was 50.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM B00363T belonged to a cluster comprising species of the genus Paenibacillus. The nearest relatives were P. residui MC-246T and P. senegalensis JC66T, with 93.2% and 92.8% gene sequence similarities, respectively. On the basis of its phenotypic characteristics and phylogenetic distinctivenes, strain YIM B00363T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus turpanensis sp. nov. is proposed. The type strain is YIM B00363T (= CGMCC 1.17507T = KCTC 43184T).

Antagonizing effects of curcumin against mercury-induced autophagic death and trace elements disorder by regulating PI3K/AKT and Nrf2 pathway in the spleen.

Mercury is a naturally occurring element and highly toxic to humans even at a low dosage. Curcumin is a polyphenol found in turmeric (Curcuma longa), widely used as a treatment strategy to improve antioxidant and anti-inflammatory properties. The purpose of this study was to investigate the potential protective mechanisms of curcumin in spleen damage induced by HgCl2. The mice were given curcumin by intragastric administration 2 h before HgCl2 injection for 24 h. At first, splenic transcriptome analysis showed that 3334 genes (2134 up and 1200 down) were differently expressed in HgCl2-induced spleen damage model. Notably, KEGG enrichment showed phosphatidylinositol 3-kinase (PI3K)-AKT might be a key signaling pathways in HgCl2-induced spleen damage. Furthermore, our data demonstrated that HgCl2 could induce autophagic cell death, evidenced by increases the protein expression of PI3K, AKT, LC3-II and p62 and the number of apoptotic cells. Furthermore, we found that curcumin significantly combated autophagic cell death, sodium overload and calcium leak induced by HgCl2. Simultaneously, further studies demonstrated that curcumin significantly activated nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway, and subsequent enhancing antioxidant defenses. Taken together, our data indicated that inorganic mercury could result in autophagic cell death, which may be related to the regulation of PI3K-AKT signaling cascades. Furthermore, Nrf2-mediated antioxidant defenses may be the target of curcumin to confers an adaptive survival response to resist spleen damage induced by HgCl2. The present study perfects the mechanism theory of HgCl2-induced spleen damage and provides a way for pharmacological intervention to prevent spleen injury.

Fsk and IBMX inhibit proliferation and proapoptotic of glioma stem cells via activation of cAMP signaling pathway.

OBJECTIVE: We aimed to find out the underlying mechanism of forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX) on glioma stem cells (GSCs). METHODS: The expression of cAMP-related protein CREB and pCREB as well as apoptosis-related proteins were detected through Western blot analysis. The level of proliferation and growth rate of human GSCs was measured through thiazolyl blue tetrazolium bromide assay and stem cells forming sphere assay. The apoptosis-related gene expression was measured through reverse transcription-polymerase chain reaction. RESULTS: cAMP signaling pathway was activated in GSCs with Fsk-IBMX administration. Fsk-IBMX could inhibit the proliferation as well as invasion and promote the apoptosis of U87 cells. Besides, U0126 could inhibit MAPK signaling pathway to increase the sensitivity of GSCs to cAMP signaling pathway. As a result, Fsk-IBMX combined with U0126 had more negative effect on GSCs. CONCLUSIONS: The relationship of cAMP and MAPK signaling pathway in GSCs may provide a potential therapeutic strategy in glioma.

The transcription factor MYB15 is essential for basal immunity (PTI) in Chinese wild grape.

MAIN CONCLUSION: MYB15 promoter of Vitis quinquangularis has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms. China is a center of origin for Vitis and is home to diverse wild Vitis genotypes, some of which show superior pathogen resistance, although the underlying molecular basis for this has not yet been elucidated. In the current study, we identified a transcription factor, MYB15, from the Chinese wild grape, Vitis quinquangularis, whose promoter region (pVqMYB15) was shown to be induced by basal immunity (also called PAMP-triggered immunity, PTI) triggered by flg22, following heterologous expression in Nicotiana benthamiana and homologous expression in grapevine. By analyzing the promoter structure and activity, we identified a unique 283 bp sequence that plays a key role in the activation of basal immunity. In addition, we showed that activation of the MYB15 promoter correlates with differences in the expression of MYB15 and RESVERATROL SYNTHASE (RS) induced by the flg22 elicitor. We further tested whether the MYB15 induction triggered by flg22 was consistent with MYB15 and RS expression following inoculation with Plasmopara viticola in grape (V. quinquangularis and Vitis vinifera) leaves. Mapping upstream signals, we found that calcium influx, an RboH-dependent oxidative burst, an MAPK cascade, and jasmonate and salicylic acid co-contributed to flg22-triggered pVqMYB15 activation. Our data suggest that the MYB15 promoter has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms.

Species delimitation and interspecific relationships of the endangered herb genus Notopterygium inferred from multilocus variations.

Species identification and discrimination is the basis of biodiversity research. In general, it is considered that numerous nucleotide variations (e.g., whole chloroplast genomes) can identify species with higher resolution than a few loci, e.g., partial chloroplast or nuclear gene fragments. In this study, we tested this hypothesis by sampling population genetics samples of the endangered herb genus Notopterygium. We sequenced the complete plastomes, five nuclear gene regions, three chloroplast DNA fragments, and a nuclear internal transcribed spacer (nrITS) region for 18 populations sampled throughout most of the geographic ranges of all six Notopterygium species. Species identification analysis showed that four DNA barcodes (matK, rbcL, trnS-trnG, and nrITS) and/or combinations of these markers achieved Notopterygium species discrimination at higher resolution than the general plastomes and nuclear gene sequences. In particular, nrITS had the highest discriminatory power among all of the individual markers. Molecular data sets and morphological evidence indicated that all six Notopterygium species could be reclassified unambiguously to four putative species clades. N. oviforme and N. franchetii had the closest relationship. Molecular dating showed that the origin and divergence of Notopterygium species was significantly associated with geological and climatic fluctuations during the middle of the Pliocene. In conclusion, our results suggest that a few nucleotide variations can achieve species discrimination with higher resolution than numerous plastomes and general nuclear gene fragments when discerning related Notopterygium species.

A specific allele of MYB14 in grapevine correlates with high stilbene inducibility triggered by Al3+ and UV-C radiation.

KEY MESSAGE: The structural differences of MYB14 promoter in two grapevine genotypes affect the expression of MYB14 and stilbene synthesis in response to Al3+ and UV-C radiation. Grapevines provide an important fruit crop worldwide, but production is often limited by pathogen infection. Stilbenes, a class of secondary metabolite, represent phytoalexins that contribute to defence against pathogens in many plants, including grapevine. It is known that the transcription factors MYB14 and MYB15 are required for the activation of the promoters of resveratrol synthase to regulate stilbene biosynthesis. In the current study, we observed that stilbene levels were more highly induced by Al3+ and UV-C radiation treatments in the cultivar Vitis labrusca 'Concord' than in the cultivar V. vinifera 'Cabernet Sauvignon'. We investigated whether genetic/structural variations in the MYB14 and MYB15 promoters between these two representative genotypes are responsible for the differences in stilbene accumulation. Significant differences in the structure and activity of the promoter of MYB14, but not MYB15 were identified between the two genotypes, following heterologous expression in Nicotiana benthamiana system and treatments with Al3+ and UV-C. Hydrogen peroxide (H2O2) was detected in Concord soon after the stress treatments, but after diphenyleneiodonium chloride pre-treatment, the expressing level of VlMYB14, the promoter activity of VlMYB14 and the accumulation of stilbenes was significantly reduced. A model is presented where the induction of MYB14 contributes to stilbene accumulation in Concord following Al3+ and UV-C treatments involving reactive oxygen species (ROS) production as an early signal.

Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS).

Genomic data are a powerful tool for elucidating the processes involved in the evolution and divergence of species. The speciation and phylogenetic relationships among Chinese Juglans remain unclear. Here, we used results from phylogenomic and population genetic analyses, transcriptomics, Genotyping-By-Sequencing (GBS), and whole chloroplast genomes (Cp genome) data to infer processes of lineage formation among the five native Chinese species of the walnut genus (Juglans, Juglandaceae), a widespread, economically important group. We found that the processes of isolation generated diversity during glaciations, but that the recent range expansion of J. regia, probably from multiple refugia, led to hybrid formation both within and between sections of the genus. In southern China, human dispersal of J. regia brought it into contact with J. sigillata, which we determined to be an ecotype of J. regia that is now maintained as a landrace. In northern China, walnut hybridized with a distinct lineage of J. mandshurica to form J. hopeiensis, a controversial taxon (considered threatened) that our data indicate is a horticultural variety. Comparisons among whole chloroplast genomes and nuclear transcriptome analyses provided conflicting evidence for the timing of the divergence of Chinese Juglans taxa. J. cathayensis and J. mandshurica are poorly differentiated based our genomic data. Reconstruction of Juglans evolutionary history indicate that episodes of climatic variation over the past 4.5 to 33.80 million years, associated with glacial advances and retreats and population isolation, have shaped Chinese walnut demography and evolution, even in the presence of gene flow and introgression.

Comparative Analyses of Chloroplast Genomes of Cucurbitaceae Species: Lights into Selective Pressures and Phylogenetic Relationships.

Cucurbitaceae is the fourth most important economic plant family with creeping herbaceous species mainly distributed in tropical and subtropical regions. Here, we described and compared the complete chloroplast genome sequences of ten representative species from Cucurbitaceae. The lengths of the ten complete chloroplast genomes ranged from 155,293 bp (C. sativus) to 158,844 bp (M. charantia), and they shared the most common genomic features. 618 repeats of three categories and 813 microsatellites were found. Sequence divergence analysis showed that the coding and IR regions were highly conserved. Three protein-coding genes (accD, clpP, and matK) were under selection and their coding proteins often have functions in chloroplast protein synthesis, gene transcription, energy transformation, and plant development. An unconventional translation initiation codon of psbL gene was found and provided evidence for RNA editing. Applying BI and ML methods, phylogenetic analysis strongly supported the position of Gomphogyne, Hemsleya, and Gynostemma as the relatively original lineage in Cucurbitaceae. This study suggested that the complete chloroplast genome sequences were useful for phylogenetic studies. It would also determine potential molecular markers and candidate DNA barcodes for coming studies and enrich the valuable complete chloroplast genome resources of Cucurbitaceae.

Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China.

The extreme conditions (e.g., cold, low oxygen, and strong ultraviolet radiation) of the high mountains provide an ideal natural laboratory for studies on speciation and the adaptive evolution of organisms. Up to now, few genome/transcriptome-based studies have been carried out on how plants adapt to conditions at extremely high altitudes. Notopterygium incisum and Notopterygium franchetii (Notopterygium, Apiaceae) are two endangered high-alpine herbal plants endemic to China. To explore the molecular genetic mechanisms of adaptation to high altitudes, we performed high-throughput RNA sequencing (RNA-seq) to characterize the transcriptomes of the two species. In total, more than 130 million sequence reads, 81,446 and 63,153 unigenes with total lengths of 86,924,837 and 62,615,693 bp, were generated for the two herbal species, respectively. OrthoMCL analysis identified 6375 single-copy orthologous genes between N. incisum and N. franchetii. In total, 381 positively-selected candidate genes were identified for both plants by using estimations of the non-synonymous to synonymous substitution rate. At least 18 of these genes potentially participate in RNA splicing, DNA repair, glutathione metabolism and the plant-pathogen interaction pathway, which were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains. Meanwhile, we detected various transcription factors that regulated the material and energy metabolism in N. incisum and N. franchetii, which probably play vital roles in the tolerance to stress in surroundings. In addition, 60 primer pairs based on orthologous microsatellite-containing sequences between the both Notopterygium species were determined. Finally, 17 polymorphic microsatellite markers (SSR) were successfully characterized for the two endangered species. Based on these candidate orthologous and SSR markers, we detected that the adaptive evolution and species divergence of N. incisum and N. franchetii were significantly associated with the extremely heterogeneous environments and climatic oscillations in high-altitude areas. This work provides important insights into the molecular mechanisms of adaptation to high-altitudes in alpine herbal plants.

De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

The anti-atherosclerotic effect of tanshinol borneol ester using fecal metabolomics based on liquid chromatography-mass spectrometry.

Tanshinol borneol ester (DBZ) is a novel experimental compound that consists of two chemical structural units from danshensu and borneol. It exhibits efficacious anti-ischemic and anti-atherosclerosis activities in rats. A fecal metabolomics based on Liquid Chromatography-Mass Spectrometry combined with clinical histopathology and blood lipid estimation was employed to assess the efficacy and the metabolic changes caused by administration of DBZ in atherosclerotic rats. There were the typical pathological features of atherosclerosis and significantly increased levels of TC, TG and LDL-C in the atherosclerotic rat group. Nevertheless, atherosclerotic rats administered both DBZ (at a dose of 40 mg kg(-1)) and simvastatin (at a dose of 20 mg kg(-1)) showed good therapeutic effects. The results of the metabolomics studies showed that 55 differential metabolites such as sebacic acid, enterodiol, nonanedioic acid, dodecanedioic acid, cholic acid, 13(S)-HPODE, deoxycholic acid, some phosphatidylglycerol and phosphatidic acids were found, indicating that abnormal metabolism occurred in the pathways of fatty acid oxidation, linoleic acid metabolism, bile acid biosynthesis and glycerophospholipid metabolism in atherosclerotic rats. Compared to those in the model group, the contents of 41 differential metabolites showed a tendency to recover to a healthy level after DBZ administration. Metabolomics studies suggested that DBZ exhibited good treatment efficacy against atherosclerosis by adjusting disturbed metabolic pathways related to atherosclerosis. This study could provide an experimental basis for DBZ's application to act as a candidate drug with anti-atherosclerosis activity.

Transcriptome Sequencing and Development of Genic SSR Markers of an Endangered Chinese Endemic Genus Dipteronia Oliver (Aceraceae).

Dipteronia Oliver (Aceraceae) is an endangered Chinese endemic genus consisting of two living species, Dipteronia sinensis and Dipteronia dyeriana. However, studies on the population genetics and evolutionary analyses of Dipteronia have been hindered by limited genomic resources and genetic markers. Here, the generation, de novo assembly and annotation of transcriptome datasets, and a large set of microsatellite or simple sequence repeat (SSR) markers derived from Dipteronia have been described. After Illumina pair-end sequencing, approximately 93.2 million reads were generated and assembled to yield a total of 99,358 unigenes. A majority of these unigenes (53%, 52,789) had at least one blast hit against the public protein databases. Further, 12,377 SSR loci were detected and 4179 primer pairs were designed for experimental validation. Of these 4179 primer pairs, 435 primer pairs were randomly selected to test polymorphism. Our results show that products from 132 primer pairs were polymorphic, in which 97 polymorphic SSR markers were further selected to analyze the genetic diversity of 10 natural populations of Dipteronia. The identification of SSR markers during our research will provide the much valuable data for population genetic analyses and evolutionary studies in Dipteronia.

Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher(®)-G constructs in regenerative medicine.

The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher(®)-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher(®)-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher(®)-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher(®)-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays.