Thirteen complete chloroplast genomes of the costaceae family: insights into genome structure, selective pressure and phylogenetic relationships.

BACKGROUND: Costaceae, commonly known as the spiral ginger family, consists of approximately 120 species distributed in the tropical regions of South America, Africa, and Southeast Asia, of which some species have important ornamental, medicinal and ecological values. Previous studies on the phylogenetic and taxonomic of Costaceae by using nuclear internal transcribed spacer (ITS) and chloroplast genome fragments data had low resolutions. Additionally, the structures, variations and molecular evolution of complete chloroplast genomes in Costaceae still remain unclear. Herein, a total of 13 complete chloroplast genomes of Costaceae including 8 newly sequenced and 5 from the NCBI GenBank database, representing all three distribution regions of this family, were comprehensively analyzed for comparative genomics and phylogenetic relationships. RESULT: The 13 complete chloroplast genomes of Costaceae possessed typical quadripartite structures with lengths from 166,360 to 168,966 bp, comprising a large single copy (LSC, 90,802 - 92,189 bp), a small single copy (SSC, 18,363 - 20,124 bp) and a pair of inverted repeats (IRs, 27,982 - 29,203 bp). These genomes coded 111 - 113 different genes, including 79 protein-coding genes, 4 rRNA genes and 28 - 30 tRNAs genes. The gene orders, gene contents, amino acid frequencies and codon usage within Costaceae were highly conservative, but several variations in intron loss, long repeats, simple sequence repeats (SSRs) and gene expansion on the IR/SC boundaries were also found among these 13 genomes. Comparative genomics within Costaceae identified five highly divergent regions including ndhF, ycf1-D2, ccsA-ndhD, rps15-ycf1-D2 and rpl16-exon2-rpl16-exon1. Five combined DNA regions (ycf1-D2 + ndhF, ccsA-ndhD + rps15-ycf1-D2, rps15-ycf1-D2 + rpl16-exon2-rpl16-exon1, ccsA-ndhD + rpl16-exon2-rpl16-exon1, and ccsA-ndhD + rps15-ycf1-D2 + rpl16-exon2-rpl16-exon1) could be used as potential markers for future phylogenetic analyses and species identification in Costaceae. Positive selection was found in eight protein-coding genes, including cemA, clpP, ndhA, ndhF, petB, psbD, rps12 and ycf1. Maximum likelihood and Bayesian phylogenetic trees using chloroplast genome sequences consistently revealed identical tree topologies with high supports between species of Costaceae. Three clades were divided within Costaceae, including the Asian clade, Costus clade and South American clade. Tapeinochilos was a sister of Hellenia, and Parahellenia was a sister to the cluster of Tapeinochilos + Hellenia with strong support in the Asian clade. The results of molecular dating showed that the crown age of Costaceae was about 30.5 Mya (95% HPD: 14.9 - 49.3 Mya), and then started to diverge into the Costus clade and Asian clade around 23.8 Mya (95% HPD: 10.1 - 41.5 Mya). The Asian clade diverged into Hellenia and Parahellenia at approximately 10.7 Mya (95% HPD: 3.5 - 25.1 Mya). CONCLUSION: The complete chloroplast genomes can resolve the phylogenetic relationships of Costaceae and provide new insights into genome structures, variations and evolution. The identified DNA divergent regions would be useful for species identification and phylogenetic inference in Costaceae.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-ß (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-ß1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Bartonella Prevalence and Genome Sequences in Rodents in Some Regions of Xinjiang, China.

In this study, we investigated Bartonella infection and its genetic diversity in rodents in Beitun, Xinjiang Uygur Autonomous Region, China. Small mammals were captured using snap traps at four sampling sites in 2018. Spleen and liver tissues were collected and cultured to isolate Bartonella strains. Whole-genome sequencing was performed on the strains identified as Bartonella by gltA gene PCR, and the average nucleotide identity (ANI) of the genomes was calculated by using FastANI v1.33. Phylogenetic trees were constructed for the samples positive for Bartonella spp. by the gltA PCR assay based on 1,290-bp gltA genes, 2,903-bp rpoB genes, and core-genome single nucleotide polymorphisms (SNPs). Among 66 rodents, 11 were positive for Bartonella, with an infection rate of 16.67%. The rodent infection rates in different tissues (χ2 = 2.133; P = 0.242), species (χ2 = 9.631; P = 0.141), and habitats (χ2 = 4.309; P = 0.312) did not show statistical differences. Bartonella spp. isolated from the rodents were phylogenetically divided into six clades (two different Bartonella species were detected in two rodents). By comparing phylogenetic trees based on gltA genes, rpoB genes, and SNPs, we found that the topological structures of several evolutionary trees are different. However, the Bartonella strains isolated in this study were clustered into six clusters in different phylogenetic trees. Broad distributions and high genetic diversity of Bartonella strains were observed among rodents in Beitun, Xinjiang. IMPORTANCE Rodent-borne Bartonella species have been associated with zoonotic diseases. Bartonella species such as Bartonella elizabethae, Bartonella grahamii, and Bartonella tribocorum can cause disease in humans. Humans can be infected by blood-sucking arthropods through the scratches and bites of an infected reservoir host or via contact with infectious rodents. Xinjiang is one of the provinces with the most abundant species of Bartonella in China, but there are few reports about the prevalence of Bartonella in the Beitun area. This research aims to investigate the occurrence and prevalence of Bartonella infection in rodents at these sampling sites and provide a basis for the prevention and control of rodent Bartonella species in Beitun and the surrounding areas of Xinjiang.

Doped nanomaterial facilitates 3D printing target plate for rapid detection of alkaloids in laser desorption/ionization mass spectrometry.

This work aims to rapidly detect toxic alkaloids in traditional Chinese medicines (TCM) using laser desorption ionization mass spectrometry (LDI-MS). We systematically investigated twelve nanomaterials (NMs) as matrices and found that MoS2 and defect-rich-WO3 (D-WO3) were the best NMs for alkaloid detection. MoS2 and D-WO3 can be used directly as matrices dipped onto conventional ground steel target plates. Additionally, they can be conveniently fabricated as three-dimensional (3D) NM plates, where the MoS2 or D-WO3 NM is doped into resin and formed using a 3D printing process. We obtained good quantification of alkaloids using a chemothermal compound as an internal standard and detected related alkaloids in TCM extracts, Fuzi (Aconiti Lateralis Radix Praeparata), Caowu (Aconiti Kusnezoffii Radix), Chuanwu (Aconiti Radix), and Houpo (Magnoliae Officinalis Cortex). The work enabled the advantageous "dip and measure" method, demonstrating a simple and fast LDI-MS approach that achieves clean backgrounds for alkaloid detection. The 3D NM plates also facilitated mass spectrometry imaging of alkaloids in TCMs. This method has potential practical applications in medicine and food safety. Doped nanomaterial facilitates 3D printing target plate for rapid detection of alkaloids in laser desorption/ionization mass spectrometry.

Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue.

Ovarian tissue cryopreservation is an effective fertility protective strategy for preadolescent female cancer patients, whose tumor treatment cannot be delayed. In the present study, the effects of sericin, as an antioxidant, on mice ovarian tissue freezing and thawing were investigated. Mice ovarian tissues were cryopreserved and thawed in medium containing 0.5% or 1%sericin (w/v), and 0.1 mM melatonin. Then, the follicular morphology was observed. The levels of antioxidant enzymes were determined, including glutathione (GSH), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and catalase (CAT). Moreover, the levels of nitric oxide (NO), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were also tested. Besides, apoptosis-related proteins Bcl-2 and Bax were determined. Our results showed that 1% sericin maintained follicular morphology, inhibited apoptosis, decreased MDA and NO levels, and boosted endogenous antioxidant enzyme levels, while had no significant effect on LDH levels. Furthermore, these effects may be related with the activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of Rapamycin (mTOR) signaling pathway, as demonstrated by increased PI3K, p-AKT and mTOR levels. These findings demonstrate that 1% sericin may reduce oxidative stress and protect ovarian tissues during freezing and thawing via PI3K/AKT/mTOR signaling pathway.

Comparative Chloroplast Genomics of 21 Species in Zingiberales with Implications for Their Phylogenetic Relationships and Molecular Dating.

Zingiberales includes eight families and more than 2600 species, with many species having important economic and ecological value. However, the backbone phylogenetic relationships of Zingiberales still remain controversial, as demonstrated in previous studies, and molecular dating based on chloroplast genomes has not been comprehensively studied for the whole order. Herein, 22 complete chloroplast genomes from 21 species in Zingiberales were sequenced, assembled, and analyzed. These 22 genomes displayed typical quadripartite structures, which ranged from 161,303 bp to 163,979 bp in length and contained 111-112 different genes. The genome structures, gene contents, simple sequence repeats, long repeats, and codon usage were highly conserved, with slight differences among these genomes. Further comparative analysis of the 111 complete chloroplast genomes of Zingiberales, including 22 newly sequenced ones and the remaining ones from the national center for biotechnology information (NCBI) database, identified three highly divergent regions comprising ccsA, psaC, and psaC-ndhE. Maximum likelihood and Bayesian inference phylogenetic analyses based on chloroplast genome sequences found identical topological structures and identified a strongly supported backbone of phylogenetic relationships. Cannaceae was sister to Marantaceae, forming a clade that was collectively sister to the clade of (Costaceae, Zingiberaceae) with strong support (bootstrap (BS) = 100%, and posterior probability (PP) = 0.99-1.0); Heliconiaceae was sister to the clade of (Lowiaceae, Strelitziaceae), then collectively sister to Musaceae with strong support (BS = 94-100%, and PP = 0.93-1.0); the clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)) was sister to the clade of (Musaceae, (Heliconiaceae, (Lowiaceae, Strelitziaceae))) with robust support (BS = 100%, and PP = 1.0). The results of divergence time estimation of Zingiberales indicated that the crown node of Zingiberales occurred approximately 85.0 Mya (95% highest posterior density (HPD) = 81.6-89.3 million years ago (Mya)), with major family-level lineages becoming from 46.8 to 80.5 Mya. These findings proved that chloroplast genomes could contribute to the study of phylogenetic relationships and molecular dating in Zingiberales, as well as provide potential molecular markers for further taxonomic and phylogenetic studies of Zingiberales.

Coordinated Patient Care via Mobile Phone-Based Telemedicine in Secondary Stroke Prevention: A Propensity Score-Matched Cohort Study.

BACKGROUND: To prevent recurrent stroke, patients need to follow evidence-based practices following discharge; however, adherence to these practices is suboptimal. PURPOSE: To evaluate whether a smartphone mobile application can improve medication adherence and stroke awareness in secondary stroke prevention. METHODS: A retrospective study design was used. Patients with ischemic stroke registered in a database between August 2018 and January 2019 were enrolled. Propensity score matching was used to match patients managed with the mobile application compared with regular practice in a 1:2 ratio. RESULTS: Sixty-five patients were paired with 123 controls. Three-month medication adherence was 93.8% in the application group versus 82.9% in the control group ( P = .036). Patients in the application group were more likely to know stroke warning signs ( P = .003) and when to call an ambulance for stroke symptoms (87.7% vs 72.4%, P = .016). CONCLUSIONS: Using a mobile application may increase medication adherence and stroke awareness in secondary stroke prevention.

[Expression of SCD1 in TFE3-rRCC and its effect on proliferation and migration].

OBJECTIVE: To investigate the expression of SCD1 in TFE3-rRCC and its effect on the proliferation and migration of TFE3-rRCC cells. METHODS: GEPIA database was used to analyze the expression level of SCD1 in different tumors and its effect on the prognosis of patients. The expression levels of SCD1 in TFE3-rRCC patients and cell lines UOK109 and UOK120 were detected by QPCR and Western blot. Liposomal shRNA was used to knock down SCD1 expression in cell lines. The changes of cell proliferation and migration ability before and after SCD1 knockdown were detected by CCK-8 and Transwell experiments. RESULTS: SCD1 expression levels were higher in all three common renal cancers, and patients with high SCD1 expression had shorter survival and worse prognosis (Logrank P<0.001). The mRNA and protein levels of SCD1 were also significantly increased in renal cancer tissues of patients with high expression of TFE3 and in TFE3-rRCC cell lines UOK109 and UOK120. After SCD1 knockdown, the proliferation and migration ability of UOK109 and UOK120 cells decreased significantly. CONCLUSION: SCD1 is highly expressed in TFE3-rRCC and can promote the proliferation and migration of TFE3-rRCC cell lines, which may be a key molecule in promoting the development of TFE3-rRCC.

[Prediction and analysis of Q-markers of Elephantopus scaber based on its UPLC fingerprint, content determination of components, and in vitro a nti-tumor activity].

This study aimed to provide scientific evidence for predicting quality markers(Q-markers) of Elephantopus scaber by establishing UPLC fingerprint of E. scaber from different geographical origins and determining the content of 13 major components, as well as conducting in vitro anti-cancer activity investigation of the main components. The chromatographic column used was Waters CORTECS UPLC C_(18)(2.1 mm×150 mm, 1.6 µm), and the mobile phase consisted of acetonitrile and 0.1% formic acid solution(gradient elution). The column temperature was set at 30 ℃, and the flow rate was 0.2 mL·min~(-1). The injection volume was 1 µL, and the detection wavelength was 240 nm. The UPLC fingerprint of E. scaber was fitted using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 edition) to determine common peaks, evaluate similarity, identify and determine the content of major components. The CCK-8 assay was used to explore the inhibitory effect of the main components on the proliferation of lung cancer cells. The results showed that in the established UPLC fingerprint of E. scaber, 35 common peaks were identified. Thirteen major components, including neochlorogenic acid(peak 1), chlorogenic acid(peak 2), cryptochlorogenic acid(peak 3), caffeic acid(peak 4), schaftoside(peak 6), galuteolin(peak 9), isochlorogenic acid B(peak 10), isochlorogenic acid A(peak 12), isochlorogenic acid C(peak 18), deoxyelephantopin(peak 28), isodeoxyelephantopin(peak 29), isoscabertopin(peak 31), and scabertopin(peak 32) were identified and quantified, and a quantitative analysis method was established. The results of the in vitro anti-cancer activity study showed that deoxyelephantopin, isodeoxyelephantopin, isoscabertopin, and scabertopin in E. scaber exhibited inhibition rates of lung cancer cell proliferation exceeding 80% at a concentration of 10 µmol·L~(-1), higher than the positive drug paclitaxel. These results indicate that the fingerprint of E. scaber is highly characteristic, and the quantitative analysis method is accurate and stable, providing references for the research on quality standards of E. scaber. Four sesquiterpene lactones in E. scaber show significant anti-cancer activity and can serve as Q-markers for E. scaber.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

Molecular evolution of chloroplast genomes in subfamily Zingiberoideae (Zingiberaceae).

BACKGROUND: Zingiberoideae is a large and diverse subfamily of the family Zingiberaceae. Four genera in subfamily Zingiberoideae each possess 50 or more species, including Globba (100), Hedychium (> 80), Kaempferia (50) and Zingiber (150). Despite the agricultural, medicinal and horticultural importance of these species, genomic resources and suitable molecular markers for them are currently sparse. RESULTS: Here, we have sequenced, assembled and analyzed ten complete chloroplast genomes from nine species of subfamily Zingiberoideae: Globba lancangensis, Globba marantina, Globba multiflora, Globba schomburgkii, Globba schomburgkii var. angustata, Hedychium coccineum, Hedychium neocarneum, Kaempferia rotunda 'Red Leaf', Kaempferia rotunda 'Silver Diamonds' and Zingiber recurvatum. These ten chloroplast genomes (size range 162,630-163,968 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 87,172-88,632 bp), a small single copy (SSC, 15,393-15,917 bp) and a pair of inverted repeats (IRs, 29,673-29,833 bp). The genomes contain 111-113 different genes, including 79 protein coding genes, 28-30 tRNAs and 4 rRNA genes. The dynamics of the genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats exhibit similarities, with slight differences observed among the ten genomes. Further comparative analysis of seventeen related Zingiberoideae species, 12 divergent hotspots are identified. Positive selection is observed in 14 protein coding genes, including accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4. Phylogenetic analyses, based on the complete chloroplast-derived single-nucleotide polymorphism data, strongly support that Globba, Hedychium, and Curcuma I + "the Kaempferia clade" consisting of Curcuma II, Kaempferia and Zingiber, form a nested evolutionary relationship in subfamily Zingiberoideae. CONCLUSIONS: Our study provides detailed information on ten complete Zingiberoideae chloroplast genomes, representing a valuable resource for future studies that seek to understand the molecular evolutionary dynamics in family Zingiberaceae. The identified divergent hotspots can be used for development of molecular markers for phylogenetic inference and species identification among closely related species within four genera of Globba, Hedychium, Kaempferia and Zingiber in subfamily Zingiberoideae.

1,10/1,11-Cyclization catalyzed by diverged plant sesquiterpene synthases is dependent on a single residue.

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.

Diterpenoids with an unprecedented ring system from Euphorbia peplus and their activities in the lysosomal-autophagy pathway.

Three novel jatrophane diterpenes, cyclojatrophanes A-C (1-3), were isolated from the seeds of Euphorbia peplus. Compounds 1-3 featured an unprecedented 5/5/5/11 tetracyclic ring system incorporating ditetrahydropyran rings. Their structures including their absolute configurations were established by extensive spectroscopic analysis, X-ray crystallographic experiments and chemical transformations. In addition, these compounds could significantly activate the lysosomal-autophagy pathway.

Accessory hepatic vein recanalization for hepatic vein-type Budd-Chiari syndrome.

PURPOSE: To explore the clinical efficacy and long-term outcomes of accessory hepatic vein (AHV) recanalization as a means of treating hepatic vein (HV)-type Budd-Chiari syndrome (BCS). METHODS: Between January 2011 and December 2018, a total of 46 symptomatic HV-type BCS patients were treated by AHV recanalization in our hospital. The technical and clinical success of this treatment, as well as associated long-term patient prognosis was assessed herein. RESULTS: The AHV recanalization approach was technically successful in 100% of patients, without any instances of complications associated with the operation. This procedure was 95.7% (44/46) clinically successful and resultant. AHV re-obstruction occurred in 12 patients. The cumulative primary one-, two-, and five-year patency rates were 77.3%, 71.7%, and 71.7%, respectively. The secondary cumulative one-, two-, and five-year patency rates were 97.7, 87.1, and 87.1%, respectively. The five-year patency rates did not differ significantly between patients treated with balloons and stents (p = .674). Based on Cox-regression analysis, younger age was an independent predictor of re-obstruction (p = .005). The cumulative one-, two-, and five-year survival rates were 97.7, 92.2, and 92.2%, respectively. CONCLUSIONS: AHV recanalization is a safe and effective treatment for HV-type BCS.

Macrocyclic diterpenoids from the seeds of Euphorbia peplus with potential activity in inducing lysosomal biogenesis.

The first phytochemical investigation of the seeds of Euphorbia peplus led to the isolation and characterization of five new (1-5), named euphopepluanones A-E, and five known diterpenoids (6-10). Their structures were established by extensive spectroscopic analysis and X-ray crystallographic experiments. Euphopepluanones A-E (1-3) feature a very rare 5/11/5-tricyclic skeleton, and euphopepluanones D-E (4-5) represent the first report of lathyrane type diterpenoids found in E. peplus. The new compounds 1-5 were assessed for their activities to induce lysosomal biogenesis through LysoTracker Red staining, in which compounds 1 and 3 could significantly induce lysosomal biogenesis. In addition, compounds 1 and 3 could promote the nuclear translocation of TFEB, a master transcriptional factor of lysosomal genes, indicating that compounds 1 and 3 induced lysosomal biogenesis through activation of TFEB.

The efflux pump inhibitor tetrandrine exhibits synergism with fluconazole or voriconazole against Candida parapsilosis.

In the last two decades, with the wide use of azoles, antifungal resistance among Candida parapsilosis has considered a matter of concern worldwide. The aim of this study is to evaluate the antifungal potentials of tetrandrine (TET) alone and in combination with fluconazole (FLC)/voriconazole (VRC) against C. parapsilosis. Susceptibility tests were performed by microdilution method, checkerboard assay, time-kill test, spot assay. Subsequently, rhodamine 6G efflux test and the expressions of transporter related genes, namely CDR1 and MDR1 for C. parapsilosis were analyzed by qRT-PCR. The susceptibility test showed that TET presented strong synergism with FLC and VRC with fractional inhibitory concentration index (FICI) in a range of 0.094-0.562. The susceptibility results were also confirmed by spot assay and time-kill studies. With TET treatment, a vast quantity of rhodamine 6G could not be pumped out from the cells as considerably intracellular red fluorescence was accumulated. Meanwhile, the expressions of efflux-associated genes presented varying degrees of inhibition. These results indicated that TET was a decent antifungal synergist to promote the antifungal efficacy of FLC/VRC, and the underlying antifungal mechanism might be associated with the inhibition of efflux pump and the elevation of intracellular drug content.

Complete Chloroplast Genome Sequences of Kaempferia Galanga and Kaempferia Elegans: Molecular Structures and Comparative Analysis.

Kaempferia galanga and Kaempferia elegans, which belong to the genus Kaempferia family Zingiberaceae, are used as valuable herbal medicine and ornamental plants, respectively. The chloroplast genomes have been used for molecular markers, species identification and phylogenetic studies. In this study, the complete chloroplast genome sequences of K. galanga and K. elegans are reported. Results show that the complete chloroplast genome of K. galanga is 163,811 bp long, having a quadripartite structure with large single copy (LSC) of 88,405 bp and a small single copy (SSC) of 15,812 bp separated by inverted repeats (IRs) of 29,797 bp. Similarly, the complete chloroplast genome of K. elegans is 163,555 bp long, having a quadripartite structure in which IRs of 29,773 bp length separates 88,020 bp of LSC and 15,989 bp of SSC. A total of 111 genes in K. galanga and 113 genes in K. elegans comprised 79 protein-coding genes and 4 ribosomal RNA (rRNA) genes, as well as 28 and 30 transfer RNA (tRNA) genes in K. galanga and K. elegans, respectively. The gene order, GC content and orientation of the two Kaempferia chloroplast genomes exhibited high similarity. The location and distribution of simple sequence repeats (SSRs) and long repeat sequences were determined. Eight highly variable regions between the two Kaempferia species were identified and 643 mutation events, including 536 single-nucleotide polymorphisms (SNPs) and 107 insertion/deletions (indels), were accurately located. Sequence divergences of the whole chloroplast genomes were calculated among related Zingiberaceae species. The phylogenetic analysis based on SNPs among eleven species strongly supported that K. galanga and K. elegans formed a cluster within Zingiberaceae. This study identified the unique characteristics of the entire K. galanga and K. elegans chloroplast genomes that contribute to our understanding of the chloroplast DNA evolution within Zingiberaceae species. It provides valuable information for phylogenetic analysis and species identification within genus Kaempferia.

The role of matrix metalloprotease (MMP) to the autolysis of sea cucumber (Stichopus japonicus).

BACKGROUND: Sea cucumber (Stichopus japonicus) is easy to autolysis in response to a variety of environmental and mechanical factors. In the current study, collagen fibres were extracted from fresh sea cucumber body wall and then incubated with endogenous matrix metalloprotease (MMP) of sea cucumber. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC), chemical analysis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis were utilized to demonstrate the changes in collagen fibres, collagen fibrils and collagen proteins. Moreover, a verification experiment was also carried out to confirm the contribution of MMP to the autolysis of sea cucumber. RESULTS: Endogenous MMP caused complete depolymerization of collagen fibres into smaller collagen fibril bundles and collagen fibrils due to the fracture of proteoglycan interfibrillar bridges. Meanwhile, endogenous MMP also caused partial degradation of collagen fibrils by releasing soluble hydroxyproline and pyridinium cross-links. Furthermore, the treatment with MMP inhibitor (1,10-phenanthroline) prevented the autolysis of tissue blocks from S. japonicus dermis. CONCLUSION: Endogenous MMP was the key enzyme in the autolysis of sea cucumber, while its action still focused on high-level structures of collagens especially collagen fibres. © 2019 Society of Chemical Industry.

Estrogen-induced miR-196a elevation promotes tumor growth and metastasis via targeting SPRED1 in breast cancer.

BACKGROUND: Estrogen plays a critical role in breast cancer (BC) progression through estrogen receptor (ER)-mediated gene regulation. Emerging studies suggest that the malignant progress of BC cells is influenced by the cross talk between microRNAs (miRNAs) and ER-α signaling. However, the mechanism and functional linkage between estrogen and miRNAs remain unclear. METHODS: The expression levels of miR-196a and SPRED1 in BC were tested by qRT-PCR in 46 paired BC and adjacent tissues and by the GEO datasets. The role of miR-196a in estrogen-induced BC development was examined by CCK-8 assay, wound healing assay, Matrigel invasion assay and tumorigenicity assay in nude mice. The binding site of ER-α in miR-196a promoter region was analyzed by ChIP-seq, ChIP assay and luciferase reporter assay. The potential targets of miR-196a in BC cells were explored using the luciferase reporter assay and western blot analysis, and the correlation between miR-196a and SPRED1 was analyzed by Spearman's correlation analysis in BC specimens and GEO dataset. TCGA BRCA data was used to characterize the ESR1 signatures according to MSigDB gene set. RESULTS: The expression levels of miR-196a were higher in ER-positive (ER+) breast tumors compared to ER-negative (ER-) tumor tissue samples. Besides, miR-196a was involved in estrogen-induced BC cell proliferation, migration and invasion. Notably, the up-regulation of miR-196a was mediated by a direct interaction with estrogen receptor α (ER-α) but not estrogen receptor ß (ER-ß) in its promoter region, and miR-196a expression levels were positively correlated to ER-α signature scores. Furthermore, SPRED1 was a new direct target of miR-196a which participated in miR-196a-promoted BC development and was suppressed by ligand-activated ER-α signal pathway. Finally, forced expression of miR-196a induced tumor growth of MCF7 cells, while inhibition of miR-196a significantly suppressed the tumor progress in vivo. CONCLUSIONS: Overall, the identification of estrogen/miR-196a/SPRED1 cascade will shed light on new molecular mechanism of estrogen signaling in BC development and therapy.

Laser capture microdissection for detecting the expression of epithelial-mesenchymal transition-related genes in epithelial and spindle cells of paraffin-embedded formalin-fixed biphasic synovial sarcoma.

Synovial sarcoma (SS) is a mesenchymal malignant neoplasm showing characteristics of epithelial-mesenchymal biphasic differentiation. SS is of uncertain cellular origin; however, studies have suggested that SS originates from a somatic stem cell population. In this study, we aim to determine whether differential morphological features of the epithelial-mesenchymal transition (EMT) contributed to the tumourigenesis of SS invasion and metastasis. Twelve paraffin-embedded formalin-fixed tissue (FFPE) SS tissue specimens were obtained, and laser capture microdissection (LCM) with the ArcturusXT system and small chip method (SCM) were used to isolate and purify spindle and epithelial cells from SS specimens. The TRIzol method was used to extract RNA, and the mRNA levels of EMT-related genes in epithelial and spindle cells of SS specimens were measured using real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results show that collection of about 2 × 104 cells from FFPE samples using LCM was sufficient for qRT-PCR, with an efficiency of 75%. Compared with LCM, 72.2% (13 of 18) RNA samples were successfully extracted using SCM to isolate cells from FFPE SS tissues. In the 16 samples (11 spindle cell samples and 5 epithelial cell samples), Snail mRNA was significantly upregulated in spindle cell areas compared with that in epithelial cell areas (P = .001). Expression levels of the epithelial marker E-cadherin and the mesenchymal marker N-cadherin were not significantly different between epithelial and spindle cell areas. In spindle cells of recurrent SS samples, the mRNA levels of E-cadherin, N-cadherin, Snail, and Slug were higher in primary SS samples than in recurrent samples. Taken together, our results indicated that in SS samples, Snail mRNA was upregulated in spindle cell areas compared with that in epithelial cell areas and that the expression of EMT-related genes was increased in primary SS. LCM could be used to isolate and purify RNA from FFPE samples.