Characterization and phylogenetic analyses of ten complete plastomes of Spiraea species.

BACKGROUND: Spiraea is a genus of deciduous shrubs that contains 80-120 species, is mainly distributed in the Northern Hemisphere and has diversified in East Asia. Spiraea species are cultivated as ornamental plants and some are used in traditional herbal medicine. Based on morphological characteristics and genetic markers, phylogenetic classification exhibits low discriminatory power. RESULTS: In present study, we assembled and characterized the chloroplast (cp) genomes of ten Spiraea species and comparatively analysed with five reported cp genomes of this genus. The cp genomes of the fifteen Spiraea species, ranging from 155,904 to 158,637 bp in length, were very conserved and no structural rearrangements occurred. A total of 85 protein-coding genes (PCGs), 37 tRNAs and 8 rRNAs were annotated. We also examined 1,010 simple sequence repeat (SSR) loci, most of which had A/T base preference. Comparative analysis of cp genome demonstrated that single copy and non-coding regions were more divergent than the inverted repeats (IRs) and coding regions and six mutational hotspots were detected. Selection pressure analysis showed that all PCGs were under purifying selection. Phylogenetic analysis based on the complete cp genome data showed that Spiraea formed a monophyletic group and was further divided into two major clades. Infrageneric classification in each clade was supported with a high resolution value. Moreover, the phylogenetic trees based on each individual mutational hotspot segment and their combined dataset also consisted of two major clades, but most of the phylogenetic relationships of interspecies were not well supported. CONCLUSIONS: Although the cp genomes of Spiraea species exhibited high conservation in genome structure, gene content and order, a large number of polymorphism sites and several mutation hotspots were identified in whole cp genomes, which might be sufficiently used as molecular markers to distinguish Spiraea species. Phylogenetic analysis based on the complete cp genome indicated that infrageneric classification in two major clades was supported with high resolution values. Therefore, the cp genome data of the genus Spiraea will be effective in resolving the phylogeny in this genus.

Comparative proteomic analysis between mature and germinating seeds in Paris polyphylla var. yunnanensis.

The long dormancy period of Paris polyphylla var. yunnanensis seeds affects the supply of this scarce plant, which is used as an important traditional Chinese medicine. Mature seeds with a globular embryo and germinating seeds with developed embryo were used to explore the mechanisms of seed germination in this species. The protein profiles between the mature and germinating seeds were compared using the isobaric tags for relative and absolute quantification (iTRAQ) approach. Of the 4,488 proteins identified, a total of 1,305 differentially expressed proteins (DEPs) were detected. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these DEPs indicated that metabolic pathways and the biosynthesis of secondary metabolites were the two top pathways. Additionally, phytohormone quantification shows that the abscisic acid (ABA) level significantly decreased, whereas the GA3 level dramatically increased among nine endogenous gibberellins (GAs), resulting in a significant increase of the GA3/ABA ratio in germinating seeds. The biosynthesis pathways of carotenoid as a precursor for ABA production and GA were further analyzed, and showed that proteinic expressions of the candidate genes in the two pathways did not correlate with the transcriptional abundances. However, 9-cis-epoxycarotenoid dioxygenase (NCED), a rate limited enzyme for ABA biosynthesis, was significantly decreased in mRNA levels in germinating seeds. By contrast, gibberellin 20-oxidase (GA20ox), a key enzyme GA biosynthesis, exhibited the major increase in one copy and a slight decrease in three others at the protentional level in germinating seeds. Gibberellin 2-oxidase (GA2ox), an inactivate enzyme in bioactive GAs, has the tendency to down-regulate in mRNA or at the proteinic level in germinating seeds. Altogether, these results suggested that the analyses of ABA and GA levels, the GA3/ABA ratio, and the expressional patterns of their regulatory genes may provide a novel mechanistic understanding of how phytohormones regulate seed germination in P. polyphylla var. yunnanensis.

Grouping of UVCB substances with dose-response transcriptomics data from human cell-based assays.

The application of in vitro biological assays as new approach methodologies (NAMs) to support grouping of UVCB (unknown or variable composition, complex reaction products, and biological materials) substances has recently been demonstrated. In addition to cell-based phenotyping as NAMs, in vitro transcriptomic profiling is used to gain deeper mechanistic understanding of biological responses to chemicals and to support grouping and read-across. However, the value of gene expression profiling for characterizing complex substances like UVCBs has not been explored. Using 141 petroleum substance extracts, we performed dose-response transcriptomic profiling in human induced pluripotent stem cell (iPSC)-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, as well as cell lines MCF7 and A375. The goal was to determine whether transcriptomic data can be used to group these UVCBs and to further characterize the molecular basis for in vitro biological responses. We found distinct transcriptional responses for petroleum substances by manufacturing class. Pathway enrichment informed interpretation of effects of substances and UVCB petroleum-class. Transcriptional activity was strongly correlated with concentration of polycyclic aromatic compounds (PAC), especially in iPSC-derived hepatocytes. Supervised analysis using transcriptomics, alone or in combination with bioactivity data collected on these same substances/cells, suggest that transcriptomics data provide useful mechanistic information, but only modest additional value for grouping. Overall, these results further demonstrate the value of NAMs for grouping of UVCBs, identify informative cell lines, and provide data that could be used for justifying selection of substances for further testing that may be required for registration.

Grouping of UVCB substances with new approach methodologies (NAMs) data.

One of the most challenging areas in regulatory science is assessment of the substances known as UVCB (unknown or variable composition, complex reaction products and biological materials). Because the inherent complexity and variability of UVCBs present considerable challenges for establishing sufficient substance similarity based on chemical characteristics or other data, we hypothesized that new approach methodologies (NAMs), including in vitro test-derived biological activity signatures to characterize substance similarity, could be used to support grouping of UVCBs. We tested 141 petroleum substances as representative UVCBs in a compendium of 15 human cell types representing a variety of tissues. Petroleum substances were assayed in dilution series to derive point of departure estimates for each cell type and phenotype. Extensive quality control measures were taken to ensure that only high-confidence in vitro data were used to determine whether current groupings of these petroleum substances, based largely on the manufacturing process and physico-chemical properties, are justifiable. We found that bioactivity data-based groupings of petroleum substances were generally consistent with the manufacturing class-based categories. We also showed that these data, especially bioactivity from human induced pluripotent stem cell (iPSC)-derived and primary cells, can be used to rank substances in a manner highly concordant with their expected in vivo hazard potential based on their chemical compositional profile. Overall, this study demonstrates that NAMs can be used to inform groupings of UVCBs, to assist in identification of repre­sentative substances in each group for testing when needed, and to fill data gaps by read-across.

The complete chloroplast genome of the traditional Chinese herb, Uncaria rhynchophylla (Rubiaceae).

Uncaria rhynchophylla is a vine plant belonging to the family Rubiaceae and has been used as medicine for a long time in China. In this study, the complete chloroplast (cp) genome sequence of U. rhynchophylla was first reported and characterized. The cp genome was 154,605 bp in length and contains a pair of inverted repeats (IRs, 34,165 bp each) separated by a large (84,327 bp) and small (12,966 bp) single-copy regions. A total of 113 unique genes were predicted, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The phylogenetic analysis suggested that U. rhynchophylla was closer to Neolamarckia cadamba.

Characterization of the complete chloroplast genome of Paederia scandens (Rubiaceae): a Chinese folk medicinal plant.

Paederia scandens has been used as a traditional medicine in Asian countries to treat jaundice, dysentery, and the pain of rheumatism. The complete chloroplast (cp) genome sequence of P. scandens was reported and characterized in this study. The cp genome is 153,626 bp in length, composed of a pair of 26,513 bp inverted repeat (IR) regions separated by a large single-copy (LSC) region of 83,712 bp and a small single-copy (SSC) region of 16,888 bp. There were 131 predicted genes (86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes) in the genome, and the overall GC content of the genome is 37.7%. Phylogenetic analysis based on the cp genome data showed that P. scandens was sister to the clade formed by Galium mollugo and G. aparine.

The complete plastid genome of Iris domestica: a traditional Chinese medicine.

Iris domestica has been used as Chinese traditional medicine to treat inflammation and throat disorders for many centuries. In this study, the complete plastid genome of I. domestica was first reported and characterized. The complete plastid genome is a typical quadripartite circular molecule of 153,729 bp in length, including a large single copy (LSC) region of 83,136 bp and a small single copy (SSC) region of 18,165 bp separated by two inverted repeat (IR) regions of 26,214 bp. A total of 132 genes including 86 protein-coding genes, 38 tRNA genes, and eight rRNA genes were identified. The phylogenetic analysis revealed that I. domestica was closer to I. gatesii.

The complete chloroplast genome sequence of a traditional Chinese medicine: Achyranthes bidentata (Amaranthaceae).

Achyranthes bidentata (Amarathaceae) has been commonly used as a traditional Chinese medicine in the treatment of osteoporosis and bone nonunion. Here, the complete chloroplast genome of A. bidentata was assembled and characterized. The cp genome is 151,451 bp in length, composed of a pair of 25,150 bp inverted repeat (IR) regions separated by a large single-copy (LSC) region of 83,899 bp and a small single-copy (SSC) region of 17,252 bp. The whole cp genome of A. bidentata contains 130 genes(85 protein-coding genes, 37 tRNAs and eight rRNAs) and the overall GC content is 36.5%. Phylogenetic analysis based on the cp genome data showed that A. bidentata was close to Cyathula capitata.

Characterization of the complete chloroplast genome of Engelhardia roxburghiana (Juglandaceae).

Engelhardia roxburghiana is a traditional Chinese medicine in the Juglandaceae family. In this study, the complete chloroplast (cp) genome sequence of E. roxburghiana was reported and characterized. The cp genome is 161,550 bp in length and contains a pair of inverted repeats (IRs, 25,862 bp) separated by a large (90,937 bp) and small (18,889 bp) single-copy regions. A total of 113 unique genes were predicted, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The phylogenetic analysis suggested that E. roxburghiana is resolved as a sister group to the clade of subfam. Juglandoideae.

Transcriptome-Wide Identification and Prediction of miRNAs and Their Targets in Paris polyphylla var. yunnanensis by High-Throughput Sequencing Analysis.

Long dormancy period of seeds limits the large-scale artificial cultivation of the scarce Paris polyphylla var. yunnanensis, an important traditional Chinese medicine. Characterizing miRNAs and their targets is crucial to understanding the role of miRNAs during seed dormancy in this species. Considering the limited genome information of this species, we first sequenced and assembled the transcriptome data of dormant seeds and their seed coats as the reference genome. A total of 146,671 unigenes with an average length of 923 bp were identified and showed functional diversity based on different annotation methods. Two small RNA libraries from respective seeds and seed coats were sequenced and the combining data indicates that 263 conserved miRNAs belonging to at least 83 families and 768 novel miRNAs in 1174 transcripts were found. The annotations of the predicted putative targets of miRNAs suggest that these miRNAs were mainly involved in the cell, metabolism and genetic information processing by direct and indirect regulation patterns in dormant seeds of P. polyphylla var. yunnanensis. Therefore, we provide the first known miRNA profiles and their targets, which will assist with further study of the molecular mechanism of seed dormancy in P. polyphylla var. yunnanensis.

PRL3-zumab, a first-in-class humanized antibody for cancer therapy.

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3+, but not PRL-3-, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3+ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become "extracellular oncotargets" that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

[Management of calyceal diverticular calculi with stenotic infundibulum by flexible ureteroscopic holmium laser infundibulectomy and lithotripsy].

OBJECTIVE: To evaluate the efficacy and safety of flexible ureteroscopic holmium laser lithotripsy in treating calyceal diverticular calculi with stenotic infundibulum and to present our initial experience. METHODS: From Nov. 2012 to Nov. 2014, 10 patients with stone-bearing calyceal diverticulum and stenotic infundibulum underwent flexible ureteroscopic holmium laser lithotripsy in our hospital, including 3 female patients and 7 male patients with an average age of 36.9 years (range: 20 to 62 years). There were 6 patients with right side while 4 patients with left side calyceal diverticular calculi. The average cumulative stone size was (1.33±0.43) cm. Five patients underwent extracorporeal shock wave lithotripsy (ESWL) before hospital admission but no stone was discharged. All the patients received intravenous urography (IVU) and CT-urography (CTU) preoperatively and underwent double-J stents placement 2 weeks before operations. A digital-fiber flexible ureteroscopy and 200 µm holmium laser fiber were used for treatment. Surgeries began with routine flexible ureteroscopy and methylene blue injection was used to identify the small ostium of infundibulum. Then infundibulectomy followed by lithotripsy was performed. All the patients receive double-J stents placement and traditional Chinese medicine for 1 to 3 months after operations. The stone clearance was estimated by kidney ureter bladder (KUB) within 3 months' follow up. RESULTS: The locations of calyceal diverticulum were upper pole in 7 patients, and interpolar regions in 4 patients. The average operation time was (123.7±59.6) min, and the average estimated blood loss was (29.3±32.1) mL. Successful flexible ureteroscopic holmium laser infundibulectomies were performed in all the 10 patients. Success rate was 100%. The stone clearance rates for 1 and 3 months after surgery were 50.0% and 80.0%, respectively, which were observed by KUB follow-up. Two patients had serious post-operative fever (>38.0 °C) in coexistence with chills. The mobidity of urosepsis was 20.0%. No major complications were identified. CONCLUSION: In selected patients, calyceal diverticular calculi with stenotic infundibulum can be treated safely and efficiently with flexible ureteroscopic homium laser lithotripsy. CTU and IVU should be completed preoperatively for calyceal diverticulum location and technique difficulty prediction. Retrograde methylene blue injection can be used to identify the ostium during surgery. And prolonged post-operation stone clearance was observed.

The relationships between chemical and genetic differentiation and environmental factors across the distribution of Erigeron breviscapus (Asteraceae).

AIMS: Erigeron breviscapus (Vant.) Hand.-Mazz. is an important, widely used Chinese herb with scutellarin, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and erigoster B being its major active compounds. We aimed to resolve the influence of biotic and abiotic factors on the concentrations of these compounds and to determine appropriate cultivation methods to improve the yields of the four compounds in this herb. METHODS: In order to detect the major genetic and natural environmental factors affecting the yields of these four compounds, we applied AFLP markers to investigate the population genetic differentiation and HPLC to measure the concentrations of four major active compounds among 23 wild populations which were located across almost the entire distribution of this species in China. The meteorological data including annual average temperature, annual average precipitation and annual average hours of sunshine were collected. The relationships among the concentrations of four compounds and environmental factors and genetic differentiation were studied. IMPORTANT FINDINGS: Low intraspecific genetic differentiation is detected, and there is no obvious correlation between the genetic differentiation and the contents of the chemical compounds. We investigated the correlation between the concentrationsof four compounds (scutellarin, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and erigoster B) and environmental factors. Concentrations of two compounds (1,5-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid) were correlated with environmental factors. The concentration of 1,5-dicaffeoylquinic acid is positively correlated with latitude, and is negatively correlated with the annual average temperature. The concentration of 3,5-dicaffeoylquinic acid is positively correlated with annual average precipitation. Therefore, changing cultivation conditions may significantly improve the yields of these two compounds. We found the concentration of scutellarin positively correlated with that of erigoster B and 3,5-dicaffeoylquinic acid, respectively. We inferred that the synthesis of these two pairs of compounds may share similar triggering mechanism as they synthesized in a common pathway.

[Protective effect of heat shock protein 70 and magnesium sulfate supplementation on renal ischemia reperfusion injury].

OBJECTIVE: To investigate whether there is a protective mechanism in exogenous magnesium ions supplement in renal ischemia reperfusion injury(IRI ), and to study the expression of heat shock protein 70 (HSP70) and HSP70-mRNA in the reperfusion injury. METHODS: A total of 90 male Japanese white rabbits (1.8-2.0 kg) were divided into three groups: ischemia reperfusion group (I-R), MgSO4 pretreatment group and sham operation group. The right kidney was cut through median abdominal incision to make solitary kidney model. The left renal artery was blocked for 1 h in I-R group and MgSO4 group. 2.5% MgSO4 1 mL/(kg×h) was given through ear vein before the artery was blocked and during the blockeage in the MgSO4 group. the kidney cortex tissue was taken 1, 2, 4, 24, 48 and 72 h after reperfusion. Immunohistochemical examination was used to determine the HSP70 expression. A modified quantitative Real time-PCR was used to quantitate HSP70-mRNA in the three groups. Pathological examination was also used to confirm the results. RESULTS: Real time-PCR showed that HSP70-mRNA began to increase at the end of 1 h, and reached the peak at the end of 2 h in both I-R and MgSO4 groups, but expression in I-R group which was remarkably higher than that in the MgSO4 group at the end of 2 h and 4 h ( P<0.05). HSP70-mRNA levels decreased rapidly at the end of 24 h. In I-R group, moderate HSP70 expression could be seen in the proximal tubules during immunohistochemical examination after reperfusion for 24 h and 48 h. By contrast, there was weak HSP70 expression in the MgSO4 group 24 h and negative 48 h after reperfusion. Epithelial shedding, border brush, inflammatory cell infiltration and protein casts were serious after 24 to 48 h reperfusion, while only slight tubular cell shedding and necrosis could be found in the MgSO4 group at the matched time. CONCLUSION: Magnesium supplement can significantly relieve the renal ischemia reperfusion injury. It can inhibit the upregulated expression of HSP70 and HSP70-mRNA in vivo, which demonstrates that the expression of HSP70 is not necessary in the protective mechanism.

[A brief history of treatment for radial head fracture with radial head prosthesis.].

Since Speed first treated the radial head fracture with a metal prosthesis in 1941, the purpose of the treatment of radial head fracture with a radial head changed from the prevention of heterotopic ossification to the prevention of proximal migration of the radius and instability of the elbow. The optimal indication is gradually determined as a non-reconstructable radial head fracture with associated injuries that would leave the elbow unstable if the radial head were resected. Types of prosthesis changed from mono-block, bipolar to modular prosthesis. Material of prosthesis changed from acrylic resin, silastic to cobalt-chrome titanium and pyrocarbon. The 60-year development indicated that the operative technology of radial head prostheses was to be improved so as to reconstruct the flexibility and stability of the elbow to meet the higher functional requirements of patents.