A comparative full-length transcriptomic resource provides insight into the perennial monocarpic mass flowering.

Perennial monocarpic mass flowering represents as a key developmental innovation in flowering time diversity in several biological and economical essential families, such as the woody bamboos and the shrubby Strobilanthes. However, molecular and genetic mechanisms underlying this important biodiversity remain poorly investigated. Here, we generated a full-length transcriptome resource incorporated into the BlueOmics database (http://blueomics.iflora.cn) for two Strobilanthes species, which feature contrasting flowering time behaviors. Using about 112 and 104 Gb Iso-seq reads together with ~185 and ~75 Gb strand-specific RNA seq data, we annotated 80 971 and 79 985 non-redundant full-length transcripts for the perennial polycarpic Strobilanthes tetrasperma and the perennial monocarpic Strobilanthes biocullata, respectively. In S. tetrasperma, we identified 8794 transcripts showing spatiotemporal expression in nine tissues. In leaves and shoot apical meristems at two developmental stages, 977 and 1121 transcripts were differentially accumulated in S. tetrasperma and S. biocullata, respectively. Interestingly, among the 33 transcription factors showing differential expression in S. tetrasperma but without differential expression in S. biocullata, three were involved potentially in the photoperiod and circadian-clock pathway of flowering time regulation (FAR1 RELATED SEQUENCE 12, FRS12; NUCLEAR FACTOR Y A1, NFYA1; PSEUDO-RESPONSE REGULATOR 5, PRR5), hence provides an important clue in deciphering the flowering diversity mechanisms. Our data serve as a key resource for further dissection of molecular and genetic mechanisms underpinning key biological innovations, here, the perennial monocarpic mass flowering.

Evolution of the FLOWERING LOCUS T-like genes in angiosperms: a core-Lamiales-specific diversification.

Plant life-history is determined by two transitions, the germination and the flowering times, in which the phosphatidylethanolamine-binding proteins (PEBP) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) play key regulatory roles. Compared to the highly conserved TFL1-likes, FT-like genes vary in copy numbers significantly in gymnosperms and monocots of the angiosperms, while sporadic duplications can be observed in eudicots. Here, via a systematic analysis of the PEBPs in angiosperms with a special focus on twelve representative species featuring high-quality genomes in the Lamiales order, we identified a successive lineage-specific but systematic expansion of FT-like genes in the families of core Lamiales. The first expansion event generated FT1-likes mainly via a core-Lamiales-specific whole-genome-duplication (cL-WGD), while on the other hand, a likely random duplication produced the FT2-likes in the lineages containing Scrophulariaceae and rest of the core Lamiales. Both FT1- and FT2-like genes were further amplified tandemly in some families. These expanded FT-likes featured highly diverged expression patterns and structural variation, indicating functional diversification. Intriguingly, some core Lamiales contained the relict MOTHER OF FT AND TFL1 like 2 (MFT2) that likely expanded in the common ancestor of angiosperms. Our data showcase the highly dynamic lineage-specific expansion of the FT-like genes, thus provide important and fresh evolutionary insights into the gene-regulatory-network underpinning flowering time diversity in Lamiales, and more generally, in angiosperms.

Plastome structure, phylogeny and evolution of plastid genes in Reevesia (Helicteroideae, Malvaceae).

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945 bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized.

Quantifying stakeholders' preference for implantable medical devices in China: a discrete choice experiment.

OBJECTIVES: This study aims to gain insight into each attribute as presented in the value of implantable medical devices, quantify attributes' strength and their relative importance, and identify the determinants of stakeholders' preferences. METHODS: A mixed-methods design was used to identify attributes and levels reflecting stakeholders' preference toward the value of implantable medical devices. This design combined literature reviewing, expert's consultation, one-on-one interactions with stakeholders, and a pilot testing. Based on the design, six attributes and their levels were settled. Among 144 hypothetical profiles, 30 optimal choice sets were developed, and healthcare professionals (decision-makers, health technology assessment experts, hospital administrators, medical doctors) and patients as stakeholders in China were surveyed. A total of 134 respondents participated in the survey. Results were analyzed by mixed logit model and conditional logit model. RESULTS: The results of the mixed logit model showed that all the six attributes had a significant impact on respondents' choices on implantable medical devices. Respondents were willing to pay the highest for medical devices that provided improvements in clinical safety, followed by increased clinical effectiveness, technology for treating severe diseases, improved implement capacity, and innovative technology (without substitutes). CONCLUSIONS: The findings of DCE will improve the current evaluation on the value of implantable medical devices in China and provide decision-makers with the relative importance of the criteria in pricing and reimbursement decision-making of implantable medical devices.

Complete plastid genome structure of 13 Asian Justicia (Acanthaceae) species: comparative genomics and phylogenetic analyses.

BACKGROUND: Justicia L. is the largest genus in Acanthaceae Juss. and widely distributed in tropical and subtropical regions of the world. Previous phylogenetic studies have proposed a general phylogenetic framework for Justicia based on several molecular markers. However, their studies were mainly focused on resolution of phylogenetic issues of Justicia in Africa, Australia and South America due to limited sampling from Asia. Additionally, although Justicia plants are of high medical and ornamental values, little research on its genetics was reported. Therefore, to improve the understanding of its genomic structure and relationships among Asian Justicia plants, we sequenced complete chloroplast (cp.) genomes of 12 Asian plants and combined with the previously published cp. genome of Justicia leptostachya Hemsl. for further comparative genomics and phylogenetic analyses. RESULTS: All the cp. genomes exhibit a typical quadripartite structure without genomic rearrangement and gene loss. Their sizes range from 148,374 to 151,739 bp, including a large single copy (LSC, 81,434-83,676 bp), a small single copy (SSC, 16,833-17,507 bp) and two inverted repeats (IR, 24,947-25,549 bp). GC contents range from 38.1 to 38.4%. All the plastomes contain 114 genes, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs. IR variation and repetitive sequences analyses both indicated that Justicia grossa C. B. Clarke is different from other Justicia species because its lengths of ndhF and ycf1 in IRs are shorter than others and it is richest in SSRs and dispersed repeats. The ycf1 gene was identified as the candidate DNA barcode for the genus Justicia. Our phylogenetic results showed that Justicia is a polyphyletic group, which is consistent with previous studies. Among them, J. grossa belongs to subtribe Tetramerinae of tribe Justicieae while the other Justicia members belong to subtribe Justiciinae. Therefore, based on morphological and molecular evidence, J. grossa should be undoubtedly recognized as a new genus. Interestingly, the evolutionary history of Justicia was discovered to be congruent with the morphology evolution. CONCLUSION: Our study not only elucidates basic features of Justicia whole plastomes, but also sheds light on interspecific relationships of Asian Justicia plants for the first time.

Complete chloroplast genomes of Sorbus sensu stricto (Rosaceae): comparative analyses and phylogenetic relationships.

BACKGROUND: Sorbus sensu stricto (Sorbus s.s.) is a genus with important economical values because of its beautiful leaves, and flowers and especially the colorful fruits. It belongs to the tribe Maleae of the family Rosaceae, and comprises about 90 species mainly distributed in China. There is on-going dispute about its infrageneric classification and species delimitation as the species are morphologically similar. With the aim of shedding light on the circumscription of taxa within the genus, phylogenetic analyses were performed using 29 Sorbus s.s. chloroplast (cp) genomes (16 newly sequenced) representing two subgenera and eight sections. RESULTS: The 16 cp genomes newly sequenced range between 159,646 bp and 160,178 bp in length. All the samples examined and 22 taxa re-annotated in Sorbus sensu lato (Sorbus s.l.) contain 113 unique genes with 19 of these duplicated in the inverted repeat (IR). Six hypervariable regions including trnR-atpA, petN-psbM, rpl32-trnL, trnH-psbA, trnT-trnL and ndhC-trnV were screened and 44-53 SSRs and 14-31 dispersed repeats were identified as potential molecular markers. Phylogenetic analyses under ML/BI indicated that Sorbus s.l. is polyphyletic, but Sorbus s.s. and the other five segregate genera, Aria, Chamaemespilus, Cormus, Micromeles and Torminalis are monophyletic. Two major clades and four sub-clades resolved with full-support within Sorbus s.s. are not consistent with the existing infrageneric classification. Two subgenera, subg. Sorbus and subg. Albocarmesinae are supported as monophyletic when S. tianschanica is transferred to subg. Albocarmesinae from subg. Sorbus and S. hupehensis var. paucijuga transferred to subg. Sorbus from subg. Albocarmesinae, respectively. The current classification at sectional level is not supported by analysis of cp genome phylogeny. CONCLUSION: Phylogenomic analyses of the cp genomes are useful for inferring phylogenetic relationships in Sorbus s.s. Though genome structure is highly conserved in the genus, hypervariable regions and repeat sequences used are the most promising molecule makers for population genetics, species delimitation and phylogenetic studies.

Antibiotic Resistance Characterization of Bacteria Isolated from Traditional Chinese Paocai.

In this work, the antibiotic resistance of 218 isolates to 9 different antibiotics was analyzed with minimum inhibitory concentration method. All Lactobacillus pentosus strains were found to be resistant to streptomycin sulfate and ciprofloxacin hydrochloride. Lactococcus lactis strains were resistant to streptomycin sulfate. Specifically, 90% Klebsiella oxytoca and all Citrobacter freundii strains were resistant to ampicillin sodium. 30% K. oxytoca strains were resistant to ciprofloxacin hydrochloride. All Bacillus albus strains were resistant to erythromycin and 80% strains were resistant to ampicillin sodium. Results from PCR analysis revealed that 90 isolates carried the aadE gene. The tetM gene was detected in four L. pentosus isolates. And the streptomycin resistant gene aadA was detected in one L. pentosus isolate. Metagenome analysis revealed that 74.7% genes associated with antibiotic resistance were antibiotic resistance genes. The tetM and aadA genes, detected in PCR analysis, were also retrieved from the paocai metagenome. In brief, this study generated the antibiotic resistance profile of some paocai-originated bacteria strains. L. pentosus found in the final edible paocai were inherently resistant to antibiotics, such as streptomycin and ciprofloxacin. Results in this work reminds us to carefully choose the LAB strains for traditional Chinese paocai production to avoid potential spreading of antibiotic resistant genes.

MicroRNA-223 protects neonatal rat cardiomyocytes and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1.

Myocardial infarction (MI), characterized by interruption of blood and oxygen to myocardium, is a common yet fatal cardiovascular event that causes progressive damage to myocardial tissue and eventually leads to heart failure. Previous studies have shown increased expression of microRNA-223 (miR-223) in infarcted myocardial tissues of humans and in rat models of MI. However, the role of miR-223 in cell survival during MI has not been elucidated. Thus, we aimed to investigate whether miR-223 participates in the regulation of cardiac ischemia-induced injury and to elucidate the underlying mechanisms of this process. qRT-PCR revealed that miR-223 expression levels are significantly upregulated in the myocardial tissues of rats with post-MI heart failure and in hypoxia-treated neonatal rat cardiomyocytes (NRCMs) and H9c2 cells, which indicates that miR-223 may be associated with chronic ischemia. We also transfected NRCMs and H9c2 cells with miR-223 mimics or inhibitors in vitro, and the results revealed that increasing miR-223 expression protected cells from hypoxia-induced apoptosis and excessive autophagy, whereas decreasing miR-223 expression had contrasting effects. Further exploration of the mechanism showed that poly(ADP-ribose) polymerase 1 (PARP-1) is a target gene of miR-223 and that silencing PARP-1 prevented hypoxia-induced cell injury; additionally, silencing PARP-1 blocked the aggravated impact of miR-223 inhibitors. Thus, PARP-1 mediates the protective effects of miR-223 in hypoxia-treated cardiomyocytes. We also investigated the involvement of the Akt/mTOR pathway in the above phenomena. We found that miR-223 overexpression and PARP-1 silencing positively regulated the Akt/mTOR pathway and that treating cells with NVP-BEZ235 (BEZ235), a novel dual Akt/mTOR inhibitor, could reverse the inhibitory effects of both the miR-223 mimics and PARP-1 siRNA on hypoxia-induced apoptosis and autophagy. Taken together, our findings showed that miR-223 protects NRCMs and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1; thus, miR-223 may be a potential target in the treatment of MI in the future.

MicroRNA-223 Regulates Cardiac Fibrosis After Myocardial Infarction by Targeting RASA1.

BACKGROUND/AIMS: Percutaneous coronary intervention reduces acute myocardial infarction (MI)-induced mortality to a great extent, but effective treatments for MI-induced cardiac fibrosis and heart failure are still lacking. MicroRNAs (miRNAs) play a variety of roles in cells and have thus been investigated extensively. MicroRNA-223 (miR-223) expression has been reported to be altered in post-MI heart failure in humans; however, the roles of miR-223 in MI remain unknown. Our study aimed to elucidate the roles of miR-223 in cardiac fibrosis. METHODS: Cultured cardiac fibroblasts (CFs) were activated by TGF-ß1 stimulation. Gain and loss of miR-223 and RAS p21 protein activator 1 (RASA1) knockdown in CFs were achieved by transfecting the cells with miR-223 mimics and inhibitors, as well as small interfering RNA-RASA1 (siRASA1), respectively. Quantitative real-time reverse transcriptase-polymerase chain reactions (qRT-PCR) was used to determine miR-223-3p and RASA1 expression levels, and Cell Counting Kit-8 (CCK-8), transwell migration and scratch assays were performed to assess CFs viability and migration, respectively. Western blotting was used to detect collagen I, collagen III, alpha-smooth muscle actin (a-SMA), RASA1, p-Akt/t-Akt, p-MEK1/2/t-MEK1/2, and p-ERK1/2/t-ERK1/2 protein expressions, and immunofluorescence assays were used to detect the expression of α-actin, vimentin and α-SMA. Luciferase assays were carried out to determine whether miR-223 binds to RASA1. Rat models of MI were established by the ligation of the left anterior descending (LAD) coronary artery. MiR-223 inhibition in vivo was achieved via intramyocardial injections of the miR-223 sponge carried by adeno-associated virus 9 (AAV9). The cardiac function was detected by echocardiography, and cardiac fibrosis was shown by Masson's trichrome staining. RESULTS: miR-223 was increased in CFs compared to cardiomypcytes, and TGF-ß1 treatment increased miR-223 expression in CFs. The miR-223 mimics enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs, while the miR-223 inhibitors had contrasting effects and partially prevented the promoting effects of TGF-ß1. qRT-PCR and western blotting revealed that miR-223 negatively regulated RASA1 expression, and the luciferase assays showed that miR-223 suppressed the luciferase activity of the RASA1 3' untranslated region (3'UTR), indicating that miR-223 binds directly to RASA1. Similar to transfection with the miR-223 mimics, RASA1 knockdown enhanced cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression in CFs. Moreover, RASA1 knockdown partially reversed the inhibitory effects of the miR-223 inhibitor on cell proliferation and migration and collagen I, collagen III, and α-SMA protein expression, indicating that the effects of miR-223 in CFs are partially mediated by the regulation of RASA1 expression. Further exploration showed that miR-223 mimics and siRASA1 promoted MEK1/2, ERK1/2 and AKT phosphorylation, while the miR-223 inhibitors had contrasting effects. The in vivo experiments confirmed the results of the in vitro experiments and showed that miR-223 inhibition prevented cardiac functional deterioration and cardiac fibrosis. CONCLUSIONS: miR-223 enhanced cell proliferation, migration, and differentiation in CFs, thus mediated cardiac fibrosis after MI partially via the involvement of RASA1.

Transcriptome analysis of the Holly mangrove Acanthus ilicifolius and its terrestrial relative, Acanthus leucostachyus, provides insights into adaptation to intertidal zones.

BACKGROUND: Acanthus is a unique genus consisting of both true mangrove and terrestrial species; thus, it represents an ideal system for studying the origin and adaptive evolution of mangrove plants to intertidal environments. However, little is known regarding the two respects of mangrove species in Acanthus. In this study, we sequenced the transcriptomes of the pooled roots and leaves tissues for a mangrove species, Acanthus ilicifolius, and its terrestrial congener, A. leucostachyus, to illustrate the origin of the mangrove species in this genus and their adaptive evolution to harsh habitats. RESULTS: We obtained 73,039 and 69,580 contigs with N50 values of 741 and 1557 bp for A. ilicifolius and A. leucostachyus, respectively. Phylogenetic analyses based on four nuclear segments and three chloroplast fragments revealed that mangroves and terrestrial species in Acanthus fell into different clades, indicating a single origin of the mangrove species in Acanthus. Based on 6634 orthologs, A. ilicifolius and A. leucostachyus were found to be highly divergent, with a peak of synonymous substitution rate (Ks) distribution of 0.145 and an estimated divergence time of approximately 16.8 million years ago (MYA). The transgression in the Early to Middle Miocene may be the major reason for the entry of the mangrove lineage of Acanthus into intertidal environments. Gene ontology (GO) classifications of the full transcriptomes did not show any apparent differences between A. ilicifolius and A. leucostachyus, suggesting the absence of gene components specific to the mangrove transcriptomes. A total of 99 genes in A. ilicifolius were identified with signals of positive selection. Twenty-three of the 99 positively selected genes (PSGs) were found to be involved in salt, heat and ultraviolet stress tolerance, seed germination and embryo development under periodic inundation. These stress-tolerance related PSGs may be crucial for the adaptation of the mangrove species in this genus to stressful marine environments and may contribute to speciation in Acanthus. CONCLUSIONS: We characterized the transcriptomes of one mangrove species of Acanthus, A. ilicifolius, and its terrestrial relative, A. leucostachyus, and provided insights into the origin of the mangrove Acanthus species and their adaptive evolution to abiotic stresses in intertidal environments.

Downregulation of tNASP inhibits proliferation through regulating cell cycle-related proteins and inactive ERK/MAPK signal pathway in renal cell carcinoma cells.

Nuclear auto-antigenic sperm protein (NASP), initially described as a highly auto-immunogenic testis and sperm-specific protein, is a histone chaperone that is proved to present in all dividing cells. NASP has two splice variants: testicular NASP (tNASP) and somatic form of NASP (sNASP). Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP. Up to now, little has been known about tNASP in renal cell carcinoma (RCC). In the present study, the molecular mechanism of tNASP in RCC was explored. The expression level of tNASP in 16 paired human RCC specimens was determined. Downregulation of tNASP by small interfering RNA (siRNA) was transfected in RCC cell lines. The effect of downregulation of tNASP by siRNA on cell colony formation and proliferation was examined by colony formation assay and CCK-8 assay, cell cycle was analyzed by flow cytometry, and the expression of cyclin D1 and P21 were detected by Western blotting. ERK/MAPK signaling was also analyzed. tNASP has a relative high expression level in human RCC tissues. Via upregulation of P21 and downregulation of cyclinD1, silence of tNASP can inhibit cell proliferation, which induces cell cycle arrest. Furthermore, ERK signaling pathway is confirmed to mediate the regulation of cell cycle-related proteins caused by silence of tNASP. Our research demonstrates that knockdown of tNASP effectively inhibits the proliferation and causes G1 phase arrest through ERK/MAPK signal pathway.

Seminal plasma protein in renal cell carcinoma: expression of semenogelin I is a predictor for cancer progression and prognosis.

The incidence of renal cell carcinoma (RCC) has been steadily rising each year. There are currently few recognized biomarkers for the diagnosis and prognosis of RCC. We investigated semenogelin I (Sg I) expression and its clinical significance in patients with RCC. The expression levels of Sg I and its protein were measured by qPCR and Western blotting, respectively. Immunohistochemistry was used to investigate the protein expression of Sg I in RCC and normal renal tissue from 53 patients. The Kaplan-Meier method and log-rank test were used to evaluate the data. By qRCR (p < 0.01) and Western blot, the level of Sg I expression in benign tissues was higher than that in RCC tissues. Expression of Sg I was observed in 30 (57 %) RCC cases, which was significantly lower than that observed in benign renal tissues from the same patients [Sg I positive in 53 (100 %) cases (p < 0.0001)] by immunohistochemistry. There was an inverse relation between Sg I expression and clinical stage (pT1-2 vs pT3-4, p < 0.0001). Patients with Sg I-negative tumor had a significantly higher risk of recurrence (Kaplan-Meier and log-rank tests, p < 0.0001). There was low Sg I expression in RCC. Sg I expression has potential value in predicting cancer progression and prognosis. These findings support the use of Sg I as a novel biomarker for RCC.

Comparative Analysis of Chloroplast Genomes for the Genus Manglietia Blume (Magnoliaceae): Molecular Structure and Phylogenetic Evolution.

Manglietia Blume, belonging to the Magnoliaceae family and mainly distributed in tropical and subtropical regions of Asia, has great scientific and economic value. In this study, we employed next-generation sequencing followed by de novo assembly to investigate the adaptive evolution of Manglietia using plastid genetic information. We newly sequenced the complete or nearly complete plastomes of four Manglietia species (Manglietia aromatica, Manglietia calcarea, Manglietia kwangtungensis, and Manglietia glauca) and conducted comparative analysis with seventeen published plastomes to examine the evolutionary pattern within this genus. The plastomes of these five newly sequenced Manglietia species range from 157,093 bp (M. calcarea2) to 160,493 bp (M. kwangtungensis), all exhibiting circular structures when mapped. Nucleotide diversity was observed across the plastomes, leading us to identify 13 mutational hotspot regions, comprising eight intergenic spacer regions and five gene regions. Our phylogenetic analyses based on 77 protein-coding genes generated phylogenetic relationships with high support and resolution for Manglietia. This genus can be divided into three clades, and the previously proposed infrageneric classifications are not supported by our studies. Furthermore, the close affinity between M. aromatica and M. calcarea is supported by the present work, and further studies are necessary to conclude the taxonomic treatment for the latter. These results provide resources for the comparative plastome, breeding, and plastid genetic engineering of Magnoliaceae and flowering plants.

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. METHODS: GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n  = 8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. RESULTS: The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. CONCLUSION: Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

Neurovascular protection of alisol A on cerebral ischemia mice through activating the AKT/GSK3ß pathway.

Alisol A, a triterpene isolated from Alisma Orientale, has been shown to exhibit anti-inflammatory effects and vascular protection. This study was designed to observe the effect of alisol A on cerebral ischemia (CI)-induced neurovascular dysfunction in the hippocampus and to further explore the potential mechanisms. The results showed that alisol A treatment improved the neurological deficits and cognitive impairment of CI mice. Alisol A reduced gliosis and improved neuronal/glial metabolism. Accordingly, alisol A inhibited inflammatory factors IL-6 and IL-1ß induced by overactivation of astrocytes and microglia, thus protecting the neurovasculature. Furthermore, alisol A promoted the survival of neurons by decreasing the ratio of Bax/Bcl-2, and protected brain microvascular endothelial cells (BMECs) by upregulating the expression of ZO-1, Occludin and CD31. The phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3ß (GSK3ß) increased after treatment with alisol A. To explore the underlying mechanism, AKT was inhibited. As expected, the neurovascular protection of alisol A above was eliminated by AKT inhibition. The present study primarily suggested that alisol A could exert neurovascular protection in the hippocampus of CI mice by activating the AKT/GSK3ß pathway and may potentially be used for the treatment of CI.

The combination of Alisma and Atractylodes ameliorates cerebral ischaemia/reperfusion injury by negatively regulating astrocyte-derived exosomal miR-200a-3p/141-3p by targeting SIRT1.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Alisma and Atractylodes (AA), a classical traditional Chinese herbal decoction, may protect against cerebral ischaemia/reperfusion injury (CIRI). However, the underlying mechanism has not been characterized. Intriguingly, exosomal microRNAs (miRNAs) have been recognized as vital factors in the pharmacology of Chinese herbal decoctions. AIM OF THE STUDY: The aim of the present study was to assess whether the neuroprotective effect of AA was dependent on the efficient transfer of miRNAs via exosomes in the brain. MATERIALS AND METHODS: Bilateral common carotid artery ligation (BCAL) was used to induce transient global cerebral ischaemia/reperfusion (GCI/R) in C57BL/6 mice treated with/without AA. Neurological deficits were assessed with the modified neurological severity score (mNSS) and Morris water maze (MWM) test. Western blot (WB) analysis was used to detect the expression of sirtuin 1 (SIRT1) in the cerebral cortex. The inflammatory state was quantitatively evaluated by measuring the expression of phospho-Nuclear factor kappa B (p-NF-κB), Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) using WB analysis and glial fibrillary acidic protein (GFAP) immunohistochemical staining. The protein expression of zonula occluden-1 (ZO-1), occludin, caudin-5 and CD31 was examined by immunohistochemical staining to determine blood‒brain barrier (BBB) permeability. Exosomes were extracted from the brain interstitial space by ultracentrifugation and identified by transmission electron microscopy (TEM), WB analysis and nanoparticle tracking analysis (NTA). The origin of exosomes was clarified by measuring the specific mRNAs within exosomes via Real Time Quantitative PCR (RT‒qPCR). Differential miRNAs in exosomes were identified using microarray screening and were validated by RT‒qPCR. Exosomes were labelled with fluorescent dye (PKH26) and incubated with bEnd.3 cells, the supernatant was collected, IL-1ß/TNF-α expression was measured using enzyme-linked immunosorbent assay (ELISA), total RNA was extracted, and miR-200a-3p/141-3p expression was examined by RT‒qPCR. In addition, the levels of miR-200a-3p/141-3p in oxygen glucose deprivation/reoxygenation (OGD/R)-induced bEnd.3 cells were quantified. The direct interaction between miR-200a-3p/141-3p and the SIRT1 3' untranslated region (3'UTR) was measured by determining SIRT1 expression in bEnd.3 cells transfected with the miR-200a-3p/141-3p mimic/inhibitor. RESULTS: Severe neurological deficits and memory loss caused by GCI/R in mice was markedly ameliorated by AA treatment, particularly in the AA medium-dose group. Moreover, AA-treated GCI/R-induced mice showed significant increases in SIRT1, ZO-1, occludin, caudin-5, and CD31 expression levels and decreases in p-NF-κB, IL-1ß, TNF-α, and GFAP expression levels compared with those in untreated GCI/R-induced mice. Furthermore, we found that miR-200a-3p/141-3p was enriched in astrocyte-derived exosomes from GCI/R-induced mice and could be inhibited by treatment with a medium dose of AA. The exosomes mediated the transfer of miR-200a-3p/141-3p into bEnd.3 cells, promoted IL-1ß and TNF-α release and downregulated the expression of SIRT1. No significant changes in the levels of miR-200a-3p/141-3p were observed in OGD/R-induced bEnd.3 cells. The miR-200a-3p/141-3p mimic/inhibitor decreased/increased SIRT1 expression in bEnd.3 cells, respectively. CONCLUSION: Our findings demonstrated that AA attenuated inflammation-mediated CIRI by inhibiting astrocyte-derived exosomal miR-200a-3p/141-3p by targeting the SIRT1 gene, which provided further evidence and identified a novel regulatory mechanism for the neuroprotective effects of AA.

Cdyl2-60aa encoded by CircCDYL2 accelerates cardiomyocyte death by blocking APAF1 ubiquitination in rats.

The loss of cardiomyocytes (CMs) after myocardial infarction (MI) is widely acknowledged to initiate the development of heart failure (HF). Herein, we found that circCDYL2 (583 nt) derived from chromodomain Y-like 2 (Cdyl2) is significantly upregulated in vitro (oxygen-glucose deprivation (OGD)-treated CMs) and in vivo (failing heart post-MI) and can be translated into a polypeptide termed Cdyl2-60aa (~7 kDa) in the presence of internal ribosomal entry sites (IRES). Downregulation of circCDYL2 significantly decreased the loss of OGD-treated CMs or the infarcted area of the heart post-MI. Additionally, elevated circCDYL2 significantly accelerated CM apoptosis via Cdyl2-60aa. We then discovered that Cdyl2-60aa could stabilize protein apoptotic protease activating factor-1 (APAF1) and promote CM apoptosis; heat shock protein 70 (HSP70) mediated APAF1 degradation in CMs by ubiquitinating APAF1, which Cdyl2-60aa could competitively block. In conclusion, our work substantiated the claim that circCDYL2 could promote CM apoptosis via Cdyl2-60aa, which enhanced APAF1 stability by blocking its ubiquitination by HSP70, suggesting that it is a therapeutic target for HF post-MI in rats.

Flora diversity survey and establishment of a plant DNA barcode database of Lomas ecosystems in Peru.

Lomas formations or "fog oases" are islands of vegetation in the desert belt of the west coast of South America, with a unique vegetation composition among the world's deserts. However, plant diversity and conservation studies have long been neglected, and there exists a severe gap in plant DNA sequence information. To address the lack of DNA information, we conducted field collections and laboratory DNA sequencing to establish a DNA barcode reference library of Lomas plants from Peru. This database provides 1,207 plant specimens and 3,129 DNA barcodes data corresponding with collections from 16 Lomas locations in Peru, during 2017 and 2018. This database will facilitate both rapid species identification and basic studies on plant diversity, thereby enhancing our understanding of Lomas flora's composition and temporal variation, and providing valuable resources for conserving plant diversity and maintaining the stability of the fragile Lomas ecosystems.

Negative feedback of SNRK to circ-SNRK regulates cardiac function post-myocardial infarction.

A limited delivery of oxygen and metabolic substrate to the heart caused by myocardial infarction (MI) impairs the cardiac function, and often results in heart failure. Here, we identified a circRNA (circ-SNRK) from SNRK (sucrose nonfermenting 1-related kinase, which can increase the cardiac mitochondrial efficiency) in cardiomyocytes (CMs). Circ-SNRK can sponge the miR-33 and in turn improved the ATP synthesis via SNRK, proving the existence of circ-SNRK - miR-33 - SNRK axis. Furthermore, we found that protein NOVA1 (NOVA alternative splicing regulator 1) could accelerate the circ-SNRK formation; a cleaved peptide (~55 kDa) from SNRK enters the nucleus and blocks the cyclization of circ-SNRK via binding to NOVA1. The aforementioned negative feedback of SNRK to circ-SNRK limited the SNRK at a proper level, and inhibited the protective role of circ-SNRK in ischemic heart. In addition, our in vivo experiment indicated that the overexpression of exogenic circ-SNRK could break this loop and improves the cardiac function post-MI in rats. Together, our results demonstrated that the negative loop of circ-SNRK with SNRK regulates the energy metabolism in CMs, thus might be a potential therapeutic target for heart failure.

Comparative Analysis of the Chloroplast Genome of Cardamine hupingshanensis and Phylogenetic Study of Cardamine.

Cardamine hupingshanensis (K. M. Liu, L. B. Chen, H. F. Bai and L. H. Liu) is a perennial herbal species endemic to China with narrow distribution. It is known as an important plant for investigating the metabolism of selenium in plants because of its ability to accumulate selenium. However, the phylogenetic position of this particular species in Cardamine remains unclear. In this study, we reported the chloroplast genome (cp genome) for the species C. hupingshanensis and analyzed its position within Cardamine. The cp genome of C. hupingshanensis is 155,226 bp in length and exhibits a typical quadripartite structure: one large single copy region (LSC, 84,287 bp), one small single copy region (17,943 bp) and a pair of inverted repeat regions (IRs, 26,498 bp). Guanine-Cytosine (GC) content makes up 36.3% of the total content. The cp genome contains 111 unique genes, including 78 protein-coding genes, 29 tRNA genes and 4 rRNA genes. A total of 115 simple sequences repeats (SSRs) and 49 long repeats were identified in the genome. Comparative analyses among 17 Cardamine species identified the five most variable regions (trnH-GUG-psbA, ndhK-ndhC, trnW-CCA-trnP-UGG, rps11-rpl36 and rpl32-trnL-UAG), which could be used as molecular markers for the classification and phylogenetic analyses of various Cardamine species. Phylogenetic analyses based on 79 protein coding genes revealed that the species C. hupingshanensis is more closely related to the species C. circaeoides. This relationship is supported by their shared morphological characteristics.