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China is a hotspot of relict plant species that were once widespread throughout the Northern Hemisphere. Recent research has demonstrated that the occurrence of long-term stable refugia in the mountainous regions of central and south-western China allowed their persistence through the late Neogene climate fluctuations. One of these relict lineages is Dipteronia, an oligotypic tree genus with a fossil record extending to the Paleocene. Here, we investigated the genetic variability, demographic dynamics and diversification patterns of the two currently recognized Dipteronia species (Dipteronia sinensis and D. dyeriana). Molecular data were obtained from 45 populations of Dipteronia by genotyping three cpDNA regions, two single copy nuclear genes and 15 simple sequence repeat loci. The genetic study was combined with niche comparison analyses on the environmental space, ecological niche modeling, and landscape connectivity analysis. We found that the two Dipteronia species have highly diverged both in genetic and ecological terms. Despite the incipient speciation processes that can be observed in D. sinensis, the occurrence of long-term stable refugia and, particularly, a dispersal corridor along Daba Shan-west Qinling, likely ensured its genetic and ecological integrity to date. Our study will not only help us to understand how populations of Dipteronia species responded to the tectonic and climatic changes of the Cenozoic, but also provide insight into how Arcto-Tertiary relict plants in East Asia survived, evolved, and diversified.

Mesenchymal stem cells (MSCs) primarily secrete physiologically functional exosomes via paracrine effects that act on various adjacent and distant cells, thus exerting their therapeutic effects. In recent years, hypoxic preconditioning, as a novel MSC culture mode, has emerged as a research hotspot. Many previous studies have shown the role and underlying regulatory mechanisms of hypoxic preconditioning in various diseases, which has provided sufficient reference materials for the MSC research field. Therefore, this review summarizes the progress in application of hypoxia-preconditioned MSC-derived exosomes that substantially increases and improves the biological activity of specific molecules, such as microRNA.

Abstract Background Huazhuo Tiaozhi granule (HTG) is a herbal medicine formula widely used in clinical practice for hypolipidaemic effects. However, the molecular mechanisms underlying dyslipidaemia treatment have not been well elucidated. Results A significant reduction in the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) was observed in the serum of patients with dyslipidaemia after HTG treatment, without disruption in the levels of aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN), and creatinine (Cr). The dyslipidaemia rat model was induced by a high-fat diet and treated with Xuezhikang (0.14 g/kg/d) or HTG (9.33 g crude herb/kg/day) by gavage for 8 weeks. Body weight and liver index were markedly decreased in dyslipidaemic rats after treatment with Xuezhikang or HTG. HTG administration markedly ameliorated hyperlipidaemia by decreasing the levels of TC and LDL-C in serum and hepatic lipid accumulation. In vitro, lipid accumulation in LO2 and HepG2 cells was alleviated by serum treatment with HTG. High lactylation was observed in 198 proteins, including lactylation of histone H2B (K6), H4 (K80). Deep sequencing of microRNAs showed that miR-155-5p was significantly downregulated. Conclusions This study demonstrates that HTG is an effective and safe formula for treating dyslipidaemia, which promotes lactylation in hepatocytes, and the retardation of miR-155-5p biogenesis. Graphical abstract

Abstract Background Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE‐13) and to investigate their underlying mechanisms. Methods Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA‐repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double‐strand break (DSB) repair were measured by western blot. Results Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation‐induced inhibition of clonogenic survival in ECA109 and TE‐13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE‐13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. Conclusions These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC.

BackgroundAlzheimer's disease (AD) is a common elderly disorder characterized by cognitive decline. Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including AD. Stem cell factor (SCF) performs its biological functions by binding to and activating receptor tyrosine kinase c-Kit. We aimed to investigate the effects of SCF/c-Kit and JAK2/STAT3 on ER stress and apoptosis in AD.MethodsThe study employed L-glutamic acid (L-Glu)-treated HT22 cells as sporadic AD cell model and APP/PS1 mice as an animal model of familiar AD. SCF, c-Kit inhibitor ISCK03 or JAK2/STAT3 inhibitor WP1066 was treated to verify the effects of SCF/c-Kit and JAK2/STAT3 on ER stress and apoptosis of L-Glu-exposed HT22 cells. Cell viability was assessed by MTT. BrdU detected cell proliferation. Flow cytometry measured cell apoptosis. The expression levels of ER stress markers GRP78, PERK, CHOP, and apoptosis protein caspase3 were determined by western blot. The effect on the mRNA of ER stress markers GRP78, PERK, CHOP and apoptotic caspase3 were quantified by RT-qPCR in primary cultured hippocampal neurons from APP/PS1 transgenic mice.ResultsAdministration of SCF significantly augmented the activity and proliferation of hippocampal neuronal cells, protecting cells against L-Glu induced ER stress-associated apoptosis. Moreover, the addition of ISCK03 (c-Kit inhibitor) or WP1066 (JAK2/STAT3 inhibitor) reversed SCF effects on ER stress and apoptosis in vitro.ConclusionWe found that SCF inhibits L-Glu-induced ER stress-associated apoptosis via JAK2/STAT3 axis in HT22 hippocampal neuronal cells, as well as in primary hippocampal neurons from APP/PS1 mice, which provides valuable insights into the molecular mechanisms underlying the pathogenesis of AD and explores novel therapeutic targets for both sporadic and familial AD.

Abstract Background Retinitis pigmentosa is a rod-cone degenerative disease that induces irreversible vision loss. This study probed the protective capacity of mesenchymal stem cell-derived small EVs (MSC-EVs) on the retinas of rd10 mice and the underlying mechanism. Methods MSC-EVs were injected into the vitreous of rd10 mice at postnatal day 14 and P21; morphology and function were examined at P28. The mechanism of action was explored by using co-culture of photoreceptor cell line 661 W and microglia cell line BV2. Results Treatment with MSC-EVs increased the survival of photoreceptors and preserved their structure. Visual function, as reflected by optomotor and electroretinogram responses, was significantly enhanced in MSC-EVs-treated rd10 mice. Mechanistically, staining for Iba1, GFAP, F4/80, CD68 and CD206 showed that MSC-EVs suppressed the activation of microglial, Müller glial and macrophages. Furthermore, western blotting showed that the treatment inhibited the NF-κB pathway. RNA-seq and qPCR showed that MSC-EVs upregulated anti-inflammatory cytokines while downregulating pro-inflammatory cytokines. MSC-EVs application in vitro decreased the number of TUNEL-positive 661 W cells co-cultured with LPS-stimulated BV2, with similar impact on the cytokine expression as in vivo study. Genetic screening predicted miR-146a to be the downstream target of MSC-EVs, which was detected in MSC-EVs and upregulated in co-cultured 661 W cells and BV2 cells after MSC-EVs treatment. Upregulation of miR-146a by using its mimic decreased the expression of the transcription factor Nr4a3, and its downregulation inhibition promoted Nr4a3 expression in both 661 W and BV2 cells. Nr4a3 was further identified as the target gene of miR-146a by dual-luciferase assay. Furthermore, overexpressing miR-146a significantly decreased the expression of LPS-induced pro-inflammatory cytokines in BV2 cells. Conclusions MSC-EVs delays retinal degeneration in rd10 mice mainly by its anti-inflammatory effect via the miR-146a-Nr4a3axis. Hence, MSC-EVs may be used in the treatment of neurodegenerative diseases.

Abstract There is growing evidence supporting the implications of exosomes-shuttled microRNAs (miRs) in the phenotypes of glioblastoma stem cells (GSCs), whilst the role of exosomal miR-27b-3p remains to be established. Herein, the aim of this study was to investigate the effect of M2 tumor-associated macrophage (TAM)-derived exosomal miR-27b-3p on the function of GSCs. Clinical glioblastoma (GBM) specimens were obtained and GSCs and M2-TAMs were isolated by fluorescence-activated cell sorting (FACS), and exosomes were separated from M2-TAMs. It was observed that M2-TAM-derived exosomes promoted the stem-like properties of GSCs. Gain- and loss- of function assays were then conducted to explore the effects of exosomal miR-27b-3p and the miR-27b-3p/MLL4/PRDM1 axis on GSC phenotypes. A xenograft tumor model of GBM was further established for in vivo substantiation. Inhibition of miR-27b-3p in M2-TAMs reduced exosomal miR-27b-3p transferred into GSCs and consequently diminished GSC viability in vitro and tumor-promoting effects of GSCs in vivo. The interaction among miR-27b-3p, mixed linked leukemia 4 (MLL4), positive regulatory domain I (PRDM1) was validated by dual-luciferase and ChIP assays. MLL4 positively regulated PRDM1 expression by inducing methylation in the PRDM1 enhancer region and ultimately reduced IL-33 expression. miR-27b-3p targeted MLL4/PRDM1 to activate IL-33 and maintain the stem-like function of GSCs. In conclusion, our study elucidated that M2-TAM-derived exosomal miR-27b-3p enhanced the tumorigenicity of GSCs through the MLL4/PRDM1/IL-33 axis.

Abstract Glioblastoma (GBM) cell-derived extracellular vesicles (EVs) have been demonstrated to modulate tumor microenvironment. In the present study, we attempted to discuss the role of hsa-microRNA-27a-3p (miR-27a-3p) delivered by GBM-EVs in M2 macrophage polarization. The isolated GBM-EVs were co-cultured with macrophages. After co-culture under normoxia/hypoxia, the effect of EV-derived hsa-miR-27a-3p on GBM cell biological processes was analyzed. Additionally, the target genes of hsa-miR-27a-3p were predicted. Moreover, the binding of enhancer of zeste homologue 1 (EZH1) to lysine-specific demethylase 3A (KDM3A) promoter region and the interaction between KDM3A and connective tissue growth factor (CTGF) were analyzed. GBM mouse models were established to verify the functions of EV-derived hsa-miR-27a-3p in vivo. We found increased hsa-miR-27a-3p in GBM tissues as well as GBM-EVs, which induced M2 polarization, thus promoting proliferative, migrative and invasive potentials of GBM cells. hsa-miR-27a-3p targeted EZH1 and promoted KDM3A expression to elevate the CTGF expression. GBM-EV-delivered hsa-miR-27a-3p promoted the KDM3A-upregulated CTGF by downregulating EZH1, thereby promoting M2 macrophage polarization and development of GBM in vivo. We demonstrated that EV-derived hsa-miR-27a-3p may promote M2 macrophage polarization to induce GBM.

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.

Gynostemma microspermum C. Y. Wu et S. K. Chen is an endemic creeping herbaceous species mainly distributed in dense forests on limestone in northwestern China. Here, the complete chloroplast genome sequence of G. microspermum was obtained by Illumina pair-end sequencing. The circular complete chloroplast genome of G. microspermum is 158,692 bp in length and contains a large single copy region (87,452 bp), a small single copy region (19,068 bp) and two short inverted repeat regions (26,086 bp). The genome sequence encodes 133 genes including 87 protein-coding genes, 37 transfer RNA genes, 8 ribosomal RNA genes and 1 pseudogene. The maximum likelihood (ML) phylogeny estimation shows that G. microspermum is sister to all other analyzed species of the genus Gynostemma with high bootstrap support.

Abstract Background Skin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing. Methods In vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton’s jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant. Results The hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis. Conclusions These findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings.

The abnormally expressed long non-coding RNA (lncRNA) H19 has a crucial function in the development and progression of cardiovascular disease; however, its role in atherosclerosis is yet to be known. We aimed to examine the impacts of lncRNA H19 on atherogenesis as well as the involved mechanism. The outcomes from this research illustrated that the expression of lncRNA H19 was elevated in mouse blood and aorta with lipid-loaded macrophages and atherosclerosis. Adeno-associated virus (AAV)-mediated lncRNA H19 overexpression significantly increased the atherosclerotic plaque area in apoE−/− mice supplied with a Western diet. The upregulation of lncRNA H19 decreased the miR-146a-5p expression but increased the levels of ANGPTL4 in mouse blood and aorta and THP-1 cells. Furthermore, lncRNA H19 overexpression promoted lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-induced THP-1 macrophages. However, the knockdown of lncRNA H19 served as a protection against atherosclerosis in apoE−/− mice and lowered the accumulation of lipids in ox-LDL-induced THP-1 macrophages. lncRNA H19 promoted the expression of ANGPTL4 via competitively binding to miR-146a-5p, thus promoting lipid accumulation in atherosclerosis. These findings altogether demonstrated that lncRNA H19 facilitated the accumulation of lipid in macrophages and aggravated the progression of atherosclerosis through the miR-146a-5p/ANGPTL4 pathway. Targeting lncRNA H19 might be an auspicious therapeutic approach for preventing and treating atherosclerotic disease.

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.

Abstract Background Gliomas are common life-threatening cancers, mainly due to their aggressive nature and frequent invasiveness and long non-coding RNAs (lncRNAs) are emerging as promising molecular targets. Therefore, we explored the regulatory mechanisms underlying the putative involvement of the lncRNA PAX-interacting protein 1- antisense RNA1/ETS proto-oncogene 1/kinesin family member 14 (PAXIP1-AS1/ETS1/KIF14) axis in glioma cell invasion and angiogenesis. Methods Firstly, we identified differentially expressed lncRNA PAXIP1-AS1 as associated with glioma based on bioinformatic data. Then, validation experiments were conducted to confirm a high expression level of lncRNA PAXIP1-AS1 in glioma tissues and cells, accompanied by upregulated KIF14. We further examined the binding between lncRNA PAXIP1-AS1, KIF14 promoter activity, and transcription factor ETS1. Next, overexpression vectors and shRNAs were delivered to alter the expression of lncRNA PAXIP1-AS1, KIF14, and ETS1 to analyze their effects on glioma progression in vivo and in vitro. Results LncRNA PAXIP1-AS1 was mainly distributed in the nucleus of glioma cells. LncRNA PAXIP1-AS1 could upregulate the KIF14 promoter activity by recruiting transcription factor ETS1. Overexpression of lncRNA PAXIP1-AS1 enhanced migration, invasion, and angiogenesis of human umbilical vein endothelial cells in glioma by recruiting the transcription factor ETS1 to upregulate the expression of KIF14, which was further confirmed by accelerated tumor growth in nude mice. Conclusions The key findings of this study highlighted the potential of the lncRNA PAXIP1-AS1/ETS1/KIF14 axis as a therapeutic target for glioma treatment, due to its role in controlling the migration and invasion of glioma cells and its angiogenesis.

Abstract Background Mesenchymal stem cells (MSCs) play a significant role in cancer initiation and metastasis, sometimes by releasing exosomes that mediate cell communication by delivering microRNAs (miRNAs). This study aimed to investigate the effects of exosomal miR-133b derived from MSCs on glioma cell behaviors. Methods Microarray-based analysis identified the differentially expressed genes (DEGs) in glioma. The expression patterns of EZH2 and miR-133b along with interaction between them were clarified in glioma. The expression of miR-133b and EZH2 in glioma cells was altered to examine their functions on cell activities. Furthermore, glioma cells were co-cultured with MSC-derived exosomes treated with miR-133b mimic or inhibitor, and EZH2-over-expressing vectors or shRNA against EZH2 to characterize their effect on proliferation, invasion, and migration of glioma cells in vitro. In vivo assays were also performed to validate the in vitro findings. Results miR-133b was downregulated while EZH2 was upregulated in glioma tissues and cells. miR-133b was found to target and negatively regulate EZH2 expression. Moreover, EZH2 silencing resulted in inhibited glioma cell proliferation, invasion, and migration. Additionally, MSC-derived exosomes containing miR-133b repressed glioma cell proliferation, invasion, and migration by inhibiting EZH2 and the Wnt/β-catenin signaling pathway. Furthermore, in vivo experiments confirmed the tumor-suppressive effects of MSC-derived exosomal miR-133b on glioma development. Conclusion Collectively, the obtained results suggested that MSC-derived exosomes carrying miR-133b could attenuate glioma development via disrupting the Wnt/β-catenin signaling pathway by inhibiting EZH2, which provides a potential treatment biomarker for glioma.

Abstract Gynostemma pentaphyllum, a member of family Cucurbitaceae, is a perennial creeping herb used as a traditional medicinal plant in China. In this study, six polymorphic nSSR and four EST‐SSR primers were used to genotype 1,020 individuals in 72 wild populations of G. pentaphyllum. The genetic diversity and population structure were investigated, and ecological niche modeling was performed to reveal the evolution and demographic history of its natural populations. The results show that G. pentaphyllum has a low level of genetic diversity and high level of variation among populations because of pervasive asexual propagation, genetic drift, and long‐term habitat isolation. Results of the Mantel test demonstrate that the genetic distance and geographic distance are significantly correlated among G. pentaphyllum natural populations. The populations can be divided into two clusters on the basis of genetic structure. Asymmetrical patterns of historical gene flow were observed among the clusters. For the contemporary, almost all the bidirectional gene flow of the related pairs was symmetrical with slight differences. Recent bottlenecks were experienced by 34.72% of the studied populations. The geographic range of G. pentaphyllum continues to expand northward and eastward from Hengduan Mountains. The present distribution of G. pentaphyllum is a consequence of its complex evolution. Polyploidy in G. pentaphyllum is inferred to be polygenetic. Finally, G. pentaphyllum is a species in need of protection, so in situ and ex situ measures should be considered in the future.

Eutrema salsugineum has long been used as the model for examining salt and other abiotic stress in plants. In addition to the forward genetics approaches widely used in the lab, natural variations undoubtedly will provide a rich genetic resource for studying molecular mechanisms underlying the stress tolerance and local adaptation of this species. We used 90 resequencing whole genomes of natural populations of this species across its Asian and North American distributions to detect the selection signals for genes involved in salt and other stresses at the species-range level and local distribution. We detected selection signals for genes involved in salt and other abiotic tolerance at the species level. In addition, several cold-induced and defense genes showed selection signals due to local adaptation in North America-NE Russia or northern China, respectively. These variations and findings provide valuable resources for further deciphering genetic mechanisms underlying the stress tolerance and local adaptations of this model species.

Prunus triloba Lindl. is a small shrub species, with many varieties and a long history of cultivation. It has been widely used for landscaping and is grown as a traditional flowering tree species in northern China. In this study, we sequenced the P. triloba Lindl. chloroplast genome, which forms a circular structure comprising 158,455 bp, including a pair of inverted repeat regions (52,634 bp), a large single-copy region (86,386 bp), and a small single-copy region (19,028 bp). We annotated 131 genes, including 86 coding sequences, 8 rRNA sequences, and 37 tRNA sequences. Furthermore, a phylogenetic analysis revealed P. triloba Lindl. is closely related to Prunus pedunculata.

Past and current climatic changes have affected the demography, patterns of genetic diversity, and genetic structure of extant species. The study of these processes provides valuable information to forecast evolutionary changes and to identify conservation priorities. Here, we sequenced two functional nuclear genes and four chloroplast DNA regions for 105 samples from 21 populations of Quercus liaotungensis across its distribution range. Coalescent-based Bayesian analysis, approximate Bayesian computation (ABC), and ecological niche modeling (ENM) were integrated to investigate the genetic patterns and demographical history of this species. Association estimates including Mantel tests and multiple linear regressions were used to infer the effects of geographical and ecological factors on temporal genetic variation and diversity of this oak species. Based on multiple loci, Q. liaotungensis populations clustered into two phylogenetic groups; this grouping pattern could be the result of adaptation to habitats with different temperature and precipitation seasonality conditions. Demographical reconstructions and ENMs suggest an expansion decline trend of this species during the Quaternary climatic oscillations. Association analyses based on nuclear data indicated that intraspecific genetic differentiation of Q. liaotungensis was clearly correlated with ecological distance; specifically, the genetic diversity of this species was significantly correlated with temperature seasonality and soil pH, but negatively correlated with precipitation. Our study highlights the impact of Pleistocene climate oscillations on the demographic history of a tree species in Northern China, and suggests that climatic and soil conditions are the major factors shaping the genetic diversity and population structure of Q. liaotungensis.

Corydalis trisecta Franch. is an endemic plant found in China. In this study, we presented the first complete chloroplast genome of C. trisecta, which was assembled and characterized based on Illumina pair-end sequencing data. The complete chloroplast genome was 161,410 bp in length, with a GC content of 41.4% in total. Its structure contained a large single copy (LSC) region of 89,127 bp and a small single copy (SSC) region of 16,993 bp, which were separated by a pair of extremely inverted repeats (IRs) of 27,645 bp each, with GC content 39.8, 87.2, and 45.2%, respectively. The phylogenetic analysis indicated that C. triscta was sister to Lamprocapons spectabilis in Papaveraceae.

Fagaceae is one of the largest and economically important taxa within Fagales. Considering the incongruence among inferences from plastid and nuclear genes in the previous Fagaceae phylogeny studies, we assess the performance of plastid phylogenomics in this complex family. We sequenced and assembled four complete plastid genomes (Fagus engleriana, Quercus spinosa, Quercus aquifolioides, and Quercus glauca) using reference-guided assembly approach. All of the other 12 published plastid genomes in Fagaceae were retrieved for genomic analyses (including repeats, sequence divergence and codon usage) and phylogenetic inference. The genomic analyses reveal that plastid genomes in Fagaceae are conserved. Comparing the phylogenetic relationships of the key genera in Fagaceae inferred from different codon positions and gene function datasets, we found that the first two codon sites dataset recovered nearly all relationships and received high support. Thus, the result suggested that codon composition bias had great influence on Fagaceae phylogenetic inference. Our study not only provides basic understanding of Fagaceae plastid genomes, but also illuminates the effectiveness of plastid phylogenomics in resolving relationships of this intractable family.

Gynostemma BL., belonging to the family Cucurbitaceae, is a genus containing 17 creeping herbaceous species mainly distributed in East Asia. It can be divided into two subgenera based on different fruit morphology. Herein, we report eight complete chloroplast genome sequences of the genus Gynostemma, which were obtained by Illumina paired-end sequencing, assembly, and annotation. The length of the eight complete cp genomes ranged from 157,576 bp (G. pentaphyllum) to 158,273 bp (G. laxiflorum). Each encoded 133 genes, including 87 protein-coding genes, 37 tRNA genes, eight rRNA genes, and one pseudogene. The four types of repeated sequences had been discovered and indicated that the repeated structure for species in the Subgen. Triostellum was greater than that for species in the Subgen. Gynostemma. The percentage of variation of the eight cp genomes in different regions were calculated, which demonstrated that the coding and inverted repeats regions were highly conserved. Phylogenetic analysis based on Bayesian inference and maximum likelihood methods strongly supported the phylogenetic position of the genus Gynostemma as a member of family Cucurbitaceae. The phylogenetic relationships among the eight species were clearly resolved using the complete cp genome sequences in this study. It will also provide potential molecular markers and candidate DNA barcodes for future studies and enrich the valuable complete cp genome resources of Cucurbitaceae.

Chloroplast DNA (cpDNA) is frequently used for species demography, evolution, and species discrimination of plants. However, the lack of efficient and universal markers often brings particular challenges for genetic studies across different plant groups. In this study, chloroplast genomes from two closely related species (Quercus rubra and Castanea mollissima) in Fagaceae were compared to explore universal cpDNA markers for the Chinese oak species in Quercus subgenus Quercus, a diverse species group without sufficient molecular differentiation. With the comparison, nine and 14 plastid markers were selected as barcoding and phylogeographic candidates for the Chinese oaks. Five (psbA-trnH, matK-trnK, ycf3-trnS, matK, and ycf1) of the nine plastid candidate barcodes, with the addition of newly designed ITS and a single-copy nuclear gene (SAP), were then tested on 35 Chinese oak species employing four different barcoding approaches (genetic distance-, BLAST-, character-, and tree-based methods). The four methods showed different species identification powers with character-based method performing the best. Of the seven barcodes tested, a barcoding gap was absent in all of them across the Chinese oaks, while ITS and psbA-trnH provided the highest species resolution (30.30%) with the character- and BLAST-based methods, respectively. The six-marker combination (psbA-trnH + matK-trnK + matK + ycf1 + ITS + SAP) showed the best species resolution (84.85%) using the character-based method for barcoding the Chinese oaks. The barcoding results provided additional implications for taxonomy of the Chinese oaks in subg. Quercus, basically identifying three major infrageneric clades of the Chinese oaks (corresponding to Groups Quercus, Cerris, and Ilex) referenced to previous phylogenetic classification of Quercus. While the morphology-based allocations proposed for the Chinese oaks in subg. Quercus were challenged. A low variation rate of the chloroplast genome, and complex speciation patterns involving incomplete lineage sorting, interspecific hybridization and introgression, possibly have negative impacts on the species assignment and phylogeny of oak species.

Abies chensiensis, an endemic evergreen trees distributed in Qinling Mountains, is listed in China Species Red List as an endangered species. In this study, the complete chloroplast genome (cpDNA) sequence of Abies chensiensis was determined from Illumina pair-end sequencing data. The cpDNA is 121,329 bp in length, contains a large single copy region (LSC) of 72,843 bp and a small single copy region (SSC) of 46,126 bp, which were separated by a pair of inverted repeat (IR) regions of 1180 bp. The genome contains 120 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 35 transfer RNA genes. The overall GC content of the whole genome is 38.3%, and the corresponding values of the LSC, SSC, and IR regions are 38.8, 37.1, and 37.5%, respectively. Phylogenetic analysis based on 20 chloroplast genomes indicates that A. chensiensis is closely related to A. sibirica.

The East Himalaya-Hengduan Mountains (EH-HM) region has a high biodiversity and harbours numerous endemic alpine plants. This is probably the result of combined orographic and climate oscillations occurring since late Tertiary. Here, we determined the genetic structure and evolutionary history of alpine oak species (including Q. spinosa, Q. aquifolioides and Q. rehderiana) using both cytoplasmic-nuclear markers and ecological niche models (ENMs), and elucidated the impacts of climate oscillations and environmental heterogeneity on their population demography. Our results indicate there were mixed genetic structure and asymmetric contemporary gene flow within them. The ENMs revealed a similar demographic history for the three species expanded their ranges from the last interglacial (LIG) to the last glacial maximum (LGM), which was consistent with effective population sizes changes. Effects of genetic drift and fragmentation of habitats were responsible for the high differentiation and the lack of phylogeographic structure. Our results support that geological and climatic factors since Miocene triggered the differentiation, evolutionary origin and range shifts of the three oak species in the studied area and also emphasize that a multidisciplinary approach combining molecular markers, ENMs and population genetics can yield deep insights into diversification and evolutionary dynamics of species.

Aceraceae is a large forest tree family that comprises many economically and ecologically important species. However, because interspecific and/or intraspecific morphological variations result from frequent interspecific hybridization and introgression, it is challenging for non-taxonomists to accurately recognize and identify the tree species in Aceraceae based on a traditional approach. DNA barcoding is a powerful tool that has been proposed to accurately distinguish between species. In this study, we assessed the effectiveness of three core standard markers (matK, rbcL and ITS) plus the chloroplast locus trnS-trnG as Aceraceae barcodes. A total of 231 sequences representing 85 species in this forest family were collected. Of these four barcode markers, the discrimination power was highest for the ITS (I) region (50%) and was progressively reduced in the other three chloroplast barcodes matK (M), trnS-trnG (T) and rbcL (R); the discrimination efficiency of the ITS marker was also greater than any two-locus combination of chloroplast barcodes. However, the combinations of ITS plus single or combined chloroplast barcodes could improve species resolution significantly; T+I (90.5% resolution) and R+M+T+I (90.5% resolution) differentiated the highest portion of species in Aceraceae. Our current results show that the nuclear ITS fragment represents a more promising DNA barcode marker than the maternally inherited chloroplast barcodes. The most efficient and economical method to identify tree species in Aceraceae among single or combined DNA barcodes is the combination of T+I (90.5% resolution).

Dipteronia (order Sapindales) is an endangered genus endemic to China and has two living species, D. sinensis and D. dyeriana. The plants are closely related to the genus Acer, which is also classified in the order Sapindales. Evolutionary studies on Dipteronia have been hindered by the paucity of information on their genomes and plastids. Here, we used next generation sequencing to characterize the transcriptomes and complete chloroplast genomes of both Dipteronia species. A comparison of the transcriptomes of both species identified a total of 7,814 orthologs. Estimation of selection pressures using Ka/Ks ratios showed that only 30 of 5,435 orthologous pairs had a ratio significantly greater than 1, i.e., showing positive selection. However, 4,041 orthologs had a Ka/Ks < 0.5 (p < 0.05), suggesting that most genes had likely undergone purifying selection. Based on orthologous unigenes, 314 single copy nuclear genes were identified. Through a combination of de novo and reference guided assembly, plastid genomes were obtained; that of D. sinensis was 157,080 bp and that of D. dyeriana was 157,071 bp. Both plastid genomes encoded 87 protein coding genes, 40 tRNAs, and 8 rRNAs; no significant differences were detected in the size, gene content, and organization of the two plastomes. We used the whole chloroplast genomes to determine the phylogeny of D. sinensis and D. dyeriana and confirmed that the two species were highly divergent. Overall, our study provides comprehensive transcriptomic and chloroplast genomic resources, which will be valuable for future evolutionary studies of Dipteronia.

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.

Genetically engineered stem cells that overexpress genes encoding therapeutic products can be exploited to correct metabolic disorders by repairing and regenerating diseased organs or restoring their function. Hair follicles are readily accessible and serve as a rich source of autologous stem cells for cell-based gene therapy. Here we isolated mesenchymal stem cells from human hair follicles (HF-MSCs) and engineered them to overexpress the human insulin gene and release human insulin in a time- and dose-dependent manner in response to rapamycin. The engineered HF-MSCs retained their characteristic cell surface markers and retained their potential to differentiate into adipocytes and osteoblasts. When mice with streptozotocin-induced type 1 diabetes were engrafted with these engineered HF-MSCs, these cells expressed and released a dose of human insulin, dramatically reversed hyperglycemia, and significantly reduced death rate. Moreover, the engineered HF-MSCs did not form detectable tumors throughout the 120-day animal tests in our experiment. Our results show that HF-MSCs can be used to safely and efficiently express therapeutic transgenes and therefore show promise for cell-based gene therapy of human disease.