Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Response of Plant and Soil N, P, and N:P Stoichiometry to N Addition in China: A Meta-Analysis.

Nitrogen (N) and phosphorus (P) are key elements essential for plant growth and development. Due to fertilizer application, rapid urbanization, and fossil fuel combustion, nitrogen deposition has reached relatively high levels in China. However, there is still uncertainty regarding the response of N:P stoichiometry in plants and soil to N deposition across different ecosystems. Therefore, a meta-analysis was conducted using 845 observations from 75 studies to evaluate the response of plant and soil N and P concentrations and N to P ratios across various ecosystems to N addition. The analysis revealed that N concentration and N:P stoichiometry in plants and soil increased under N addition, while P concentration in plants and soil decreased on average. Furthermore, the magnitude of these responses was related to the N input rate and experimental duration. Finally, the effects of N addition on N concentration, P concentration, and N:P in terrestrial ecosystems would alter their allocation patterns, depending on relevant climate factors such as mean annual temperature and mean annual precipitation. This study highlights the ecological impact of N addition on the biogeochemical cycling of major elements (N and P) in terrestrial ecosystems in China. These findings are necessary for improving our understanding of the characteristics of plant ecological stoichiometry and helping to plan measures for increasing N deposition.

Enantioselective Divergent Syntheses of Cephalotaxus Alkaloids: (-)-Cephalotaxine, (-)-Cephalotine B, and (-)-Fortuneicyclidins A and B.

A new strategy focusing on the last-stage asymmetric assembly of the ring D, which inherently possesses the densest part of stereogenic centers and functional groups in the A/B/C/D ring system of (-)-cephalotaxine, has been developed, in which a novel Rh-catalyzed asymmetric (2 + 3) annulation of tertiary enamides with enoldiazoacetates is designed and explored for enantioselective construction of the crucial cyclopentane ring D bearing a unique spirocyclic aza-quaternary stereocenter. Based on the expeditious access of chiral functionalized building block with the tetracyclic A/B/C/D ring system, a concise enantioselective total synthesis of (-)-cephalotaxine starting from readily available homopiperonyl alcohol has been achieved in nine steps with only two column chromatography purifications. Following the tactical introduction of the Meinwald rearrangement, enantioselective divergent syntheses of (-)-cephalotine B with an additional C3-O-C11 oxo-bridged bond (14 steps), (-)-fortuneicyclidin B with an unprecedented C3-C10 bond (14 steps), and its 2-epimer (-)-fortuneicyclidin A (16 steps) have been also accomplished for the first time.

Iron(III)-catalyzed tandem annulation of indolyl-substituted p-quinone methides with ynamides for the synthesis of cyclopenta[b]indoles.

The unique reactivity of indolyl-substituted p-QMs as a new type of two-carbon synthon has been explored for the first time in a novel iron(III)-catalyzed tandem annulation. This (2+2) annulation/retro-4π electrocyclization/imino-Nazarov cyclization cascade reaction is characterized by an unusual structural reconstruction of indolyl-substituted p-QMs, leading to an expeditious assembly of synthetically important functionalized cyclopenta[b]indoles.

Complete mitochondrial genome of Parnassius glacialis (Lepidoptera: Papilionidae).

Parnassius glacialis is a butterfly species distributed in China, Korea, Japan. The complete P. glacialis mitochondrial genome was assembled using Illumina sequencing data. The mitogenome is 15,353 bp long and contains 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. A phylogenetic analysis of P. glacialis and 14 related Papilionidae species indicated that P. glacialis is clustered with other Parnassius species. This study generated useful genetic information for future studies on the taxonomy, phylogeny, and evolution of Papilionidae species.

The complete mitochondrial genome of Matsumuramata muiri (Hemiptera: Delphacidae).

In this study, we sequenced, annotated, and analyzed the complete mitochondrial genome of Matsumuramata muiri (Kirkaldy, 1907) (Hemiptera: Delphacidae). The mitogenome was 16,276 bp in length with high A + T content (76.28%), containing 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. Gene rearrangement of M. muiri was congruent with those of some reported delphacid species. All protein-coding genes initiated with ATN, except for nad5, which used non-canonical start codon GTG. The predicted secondary structures of all tRNA genes were typical cloverleaf except for trnS1 (AGN), lacking the dihydrouridine (DHU) stem.

The complete chloroplast genome sequence of Quercus sessilifolia Blume (Fagaceae).

Quercus sessilifolia Blume is one of the dominant tree species in East Asian evergreen broadleaved forests. In this study, we assembled and characterized the plastome of Q. sessilifolia using Illumina paired-end data. The circular genome is 160,813 bp in size, consisting of two copies of inverted repeat (IR) regions of 25,862 bp, one large single-copy (LSC) region of 90,218 bp, and one small single-copy (SSC) region of 18,871 bp. It encodes a total of 113 unique genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis based on 28 chloroplast genome sequences indicated that Q. sessilifolia was most closely related to Q. myrsinifolia with 90% bootstrap support.

Functional characterization of C-TERMINALLY ENCODED PEPTIDE (CEP) family in Brassica rapa L.

The small regulatory C-TERMINALLY ENCODED PEPTIDE (CEP) peptide family plays crucial roles in plant growth and stress response. However, little is known about this peptide family in Brassica species. Here, we performed a systematic analysis to identify the putative Brassica rapa L. CEP (BrCEP) gene family. In total, 27 BrCEP genes were identified and they were classified into four subgroups based on the CEP motifs similarity. BrCEP genes displayed distinct expression patterns in response to both developmental and several environmental signals, suggesting their broad roles during Brassica rapa development. Furthuremore, the synthetic BrCEP3 peptide accelerated Brassica rapa primary root growth in a hydrogen peroxide (H2O2) and Ca2+ dependent manner. In summary, our work will provide fundamental insights into the physiological function of CEP peptides during Brassica rapa development.

Copper-Catalyzed (4+1) Cascade Annulation of Terminal Alkynes with 2-(Tosylmethyl)anilines: Synthesis of 2,3-Disubstituted Indoles.

A novel strategy based on Cu-catalyzed (4+1) cascade annulation of terminal alkynes as one-carbon synthons with 2-(tosylmethyl)anilines has been developed for the expeditious synthesis of 2,3-disubstituted indoles, in which in situ generations of aza-o-quinone methides and alkynyl-copper(I) species are involved. This annulation provides an effective method for the assembly of synthetically and structurally interesting 2,3-disubstituted indoles.

Tandem (2 + 2) Annulation/Retro-4π Electrocyclization/Imino-Nazarov Cyclization Reaction of p-Quinone Methides with Ynamides: Expeditious Construction of Functionalized Aminoindenes.

A new tandem annulation of p-quinone methides (p-QMs) with ynamides is described. This cascade reaction features a unique combination of (2 + 2) annulation, retro-4π electrocyclization, and imino-Nazarov cyclization, wherein vinyl p-quinone methides (p-VQMs) as one of the key intermediates have been identified chemically. Significantly, an unusual structural reconstruction of p-QMs involving the cleavage of the C5-C6 bond and the late-stage formation of the C4-C6 bond is involved, leading to a methodology development for the construction of functionalized aminoindenes.

Ethylene-induced stomatal closure is mediated via MKK1/3-MPK3/6 cascade to EIN2 and EIN3.

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H2 O2 ), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H2 O2 generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3-MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H2 O2 to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.

Synergy Factorized Bilinear Network with a Dual Suppression Strategy for Brain Tumor Classification in MRI.

Automatic brain tumor classification is a practicable means of accelerating clinical diagnosis. Recently, deep convolutional neural network (CNN) training with MRI datasets has succeeded in computer-aided diagnostic (CAD) systems. To further improve the classification performance of CNNs, there is still a difficult path forward with regards to subtle discriminative details among brain tumors. We note that the existing methods heavily rely on data-driven convolutional models while overlooking what makes a class different from the others. Our study proposes to guide the network to find exact differences among similar tumor classes. We first present a "dual suppression encoding" block tailored to brain tumor MRIs, which diverges two paths from our network to refine global orderless information and local spatial representations. The aim is to use more valuable clues for correct classes by reducing the impact of negative global features and extending the attention of salient local parts. Then we introduce a "factorized bilinear encoding" layer for feature fusion. The aim is to generate compact and discriminative representations. Finally, the synergy between these two components forms a pipeline that learns in an end-to-end way. Extensive experiments exhibited superior classification performance in qualitative and quantitative evaluation on three datasets.

The complete chloroplast genome sequence of Distylium macrophyllum (Hamamelidaceae).

Distylium macrophyllum H.T.Chang is a critically endangered tree species endemic to southern China. In this study, we assembled and characterized the plastome of D. macrophyllum using Illumina paired-end data. The circular genome is 159,089 bp in size, consisting of two copies of inverted repeat (IR) regions of 26,235 bp, one large single-copy (LSC) region of 87,822 bp, and one small single-copy (SSC) region of 18,797 bp. It encodes a total of 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis based on 13 cp genome sequences indicated that D. macrophyllum was sister to the clade of D. tsiangii and Parrotia subaequalis.

UV RESISTANCE LOCUS8 mediates ultraviolet-B-induced stomatal closure in an ethylene-dependent manner.

Although the UV RESISTANCE LOCUS 8 (UVR8)-CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-ELONGATED HYPOCOTYL5 (HY5) signaling pathway, ethylene, hydrogen peroxide (H2O2), and nitric oxide (NO) all participate in ultraviolet-B (UV-B)-triggered stomatal closing, their interrelationship is not clear. Here, we found that UV-B-induced the expression of ethylene biosynthetic genes, production of ethylene, H2O2, and NO, and stomata closing were impaired in uvr8, cop1, and hy5 mutants. UV-B-induced NO production and stomata closing were also defective in mutants for ETHYLENE RESPONSE 1 (ETR1), ETHYLENE INSENSITIVE 2 (EIN2), and EIN3, but UV-B-triggered H2O2 generation was only inhibited in etr1. In either the absence or presence of UV-B, ethylene triggered H2O2 production but not NO generation and stomatal closure in cop1 and hy5, and stomata closing in cop1 and hy5 was induced by NO but not H2O2. Moreover, NO production and stomatal closure were constitutively caused by over-expression of COP1 or HY5 in ein2 and ein3, but not by over-expression of EIN2 or EIN3 in cop1 and hy5. Our data indicate that the UVR8-COP1-HY5 signaling module mediates UV-B-induced ethylene production, ethylene is then perceived by ETR1 to induce H2O2 synthesis. H2O2 induces NO generation and subsequent stomata closing via an EIN2, EIN3, COP1, and HY5-dependent pathway(s).

Comparative study of dual energy CT iodine imaging and standardized concentrations before and after chemoradiotherapy for esophageal cancer.

BACKGROUND: To compare dual energy CT iodine imaging and standardized iodine concentration before and after chemoradiotherapy (CRT) for esophageal cancer and evaluate the efficacy of CRT for EC by examining DECT iodine maps and standard CT values. METHODS: The clinical data of 45 patients confirmed by pathology with newly diagnosed esophageal cancer who underwent concurrent CRT from February 2012 to January 2017 in our department of radiology were collected. All patients underwent dual-source dual-energy CT (DECT) before and after CRT. Normalized iodine concentration (NIC) and normalized CT (NCT) corresponding to the overall cancer lesion and its maximum cross-sectional area were observed and compared. Additionally, 30 healthy individuals were compared as control group. After treatment, the patients were divided into two groups according to RECIST1.1: treatment effective group and ineffective group. RESULTS: There were 33 patients (CR 9, PR 24) in the effective group and 12 patients (SD 12, PD 0) in the ineffective group. There was no significant difference in the NIC-A, NIC-V, NCT-A and NCT-A indexes between the effective group (B group) and the ineffective group (C group) before treatment (P > 0.05). After the treatment, the above-mentioned indexes in the effective group of patients were significantly lower than before treatment, and compared with the ineffective group, the NIC-A, NIC-V, NCT-A and NCT-V values of the effective group were significantly lower than those of ineffective group (P < 0.05). After treatment, the NIC-V and NCT-V in the ineffective group were lower than before treatment, and the difference was statistically significant (P < 0.05). However, their NIC-A and NCT-A were not statistically different from those before treatment (P > 0.05). CONCLUSION: Using DECT iodine map, the changes of NIC and NIC before and after CRT in patients with esophageal cancer can evaluate the effect of CRT, and does not increase the radiation dose, so it is suitable for clinical use.

Evaluation of serum retinol-binding protein-4 levels as a biomarker of poor short-term prognosis in ischemic stroke.

The aim was to investigate the relationship between retinol-binding protein 4 (RBP4) levels and short-term functional outcome, and to determine its possible role in acute ischemic stroke (AIS). In a prospective observational study, 299 first-ever AIS who were admitted to our hospital were included. Serum levels of RBP4 were assayed and severity of stroke was evaluated with the National Institutes of Health Stroke Scale (NIHSS) score on admission. The prognostic value of RBP4 to predict the poor outcome within 3 months was compared with the NIHSS and with other known outcome predictors. The median age of the included patients was 66 (interquartile range (IQR): 55-77) years and 155 (51.8%) were women. A poor functional outcome was found in 88 patients (29.4%), and significantly higher RBP4 values were found in poor outcomes rather than good outcomes patients (P<0.001). The poor outcomes distribution across the RBP4 quartiles ranged between 9.3% (first quartile) and 60.8% (fourth quartile). In multivariate models comparing the second(Q2), third, and fourth quartiles against the first quartile of the RBP4, RBP4 in Q3 and Q4 were associated with poor functional outcome, and increased risk of poor functional outcome by 144% (OR: 2.44; 95% confidence interval (CI): 1.22-5.03) and 602% (7.02; 3.11-12.24), respectively. Interestingly, RBP4 improved the NIHSS score (area under the curve (AUC) of the combined model, 0.79; 95% CI: 0.74-0.85; P<0.001). The data showed that elevated serum levels of RBP4 at admission were associated with severity and prognosis of AIS, suggesting that vitamin A metabolism or impaired insulin signaling could be involved.

Evaluation of apparent diffusion coefficient measurements of brain injury in type 2 diabetics with retinopathy by diffusion-weighted MRI at 3.0 T.

Diabetes is often associated with impairments in brain functioning. However, the injury of specific functioning areas of the brain is not clear. To address this problem, the present study was designed to investigate possible brain functioning change in specific brain areas, particularly in areas associated with vision function, in patients with proliferative and nonproliferative diabetic retinopathy (PDR and NPDR) using the diffusion-weighted imaging technology. Conventional MRI was performed in 45 diabetic patients, 30 of whom had diabetic retinopathy (DR) involvement (half PDR, and half NPDR) and 15 of whom were diabetic patients without retinopathy and with normal ophthalmologic examination. The apparent diffusion coefficient (ADC) values were calculated in the orbitofrontal cortex (OFC), cingulated gyrus, thalamus, dorsomedial and dorsolateral frontal cortex, and corona radiate. The ADC values of the OFC, cingulated gyrus, and visual cortex were significantly increased in patients with PDR and NPDR compared with both patients without retinopathy and the control group (P<0.01). The ADC values of the OFC, cingulated gyrus, and visual cortex were significantly increased in patients with PDR compared with NPDR. The duration of disease and values of hemoglobin A1c were significantly correlated with ADC values of the OFC, cingulated gyrus, and visual cortex, respectively (P<0.01 or <0.05). We observed significantly increased ADC values of the visual center (OFC, cingulated gyrus, and visual cortex), supporting the association between DR and impairment in brain functioning. Diffusion-weighted imaging may serve to assess subclinical neurological involvement in DR, even when brain structural changes are absent.

Nutrient distribution indicated whole-tree harvesting as a possible factor restricting the sustainable productivity of a poplar plantation system in China.

We evaluated the biomass and contents of five major macronutrients (N, P, K, Ca and Mg) in 10-year-old poplar trees (Populus deltoids Bartr. cv. "Lux"), and determined their nutrient use efficiencies (NUEs) at Zhoushan Forestry Farm (32°20' N, 119°40' E), Jiangsu province, in eastern China. The above- and below-ground biomass of poplar trees was 161.7 t ha(-1), of which 53.3% was stemwood. The nutrient contents in the aboveground part were as follows: 415.1 kg N ha(-1), 29.7 kg P ha(-1), 352.0 kg K ha(-1), 1083.0 kg Ca ha(-1), and 89.8 kg Mg ha(-1). The highest nutrient contents were in stembark, followed by branches, roots, stemwood, and foliage. The NUEs of the aboveground parts of poplar for N, P, K, Ca and Mg were 0.313, 4.377, 0.369, 0.120, 1.448 t dry biomass kg(-1) nutrient, respectively, while those of stemwood were 1.294, 33.154, 1.253, 0.667, and 3.328 t dry biomass kg(-1), respectively. The cycling coefficients, defined as the percentage of annual nutrient return in annual nutrient uptake, of N, P, K, Ca and Mg for the aboveground part were 87, 95, 69, 92, and 84%, respectively. Based on the NUE and nutrient cycling characteristics, shifting from whole-tree harvesting to stemwood-only harvesting and appropriately extending the harvest rotation could prevent site deterioration and support sustainable productivity of poplar plantation systems.

Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis.

Heterotrimeric G proteins function as key players in hydrogen peroxide (H2O2) production in plant cells, but whether G proteins mediate ethylene-induced H2O2 production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H2O2 in guard cell ethylene signalling. In wild-type leaves, ethylene-triggered H2O2 synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene-induced H2O2 production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H2O2 production in response to ethylene. Ethylene-triggered H2O2 generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H2O2 rescued the defect of RAN1 and EIN4 mutants or etr1-3 in ethylene-induced H2O2 production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1-1 and etr1-9 in ethylene-induced H2O2 production. Stomata of CTR1 mutants showed constitutive H2O2 production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H2O2, but do generate H2O2 following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene-induced stomatal closure via H2O2 production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H2O2 signalling in guard cells.

The role and interactions of cytosolic alkalization and hydrogen peroxide in ultraviolet B-induced stomatal closure in Arabidopsis.

Cytosolic alkalization has been shown to function as a key player in multiple stimuli-induced stomatal closure, but its role and relationship with hydrogen peroxide (H2O2) in ultraviolet B (UV-B)-induced stomatal closure remains unknown. In this paper, by stomatal bioassay and laser-scanning confocal microscopy, we observed that 0.5 W m(-2) UV-B induced cytosolic alkalization and H2O2 production in guard cells while inducing stomatal closure in Arabidopsis (Arabidopsis thaliana). Butyrate (a weak acid) reduced the cytosolic pH/H2O2 production and prevented stomatal closure by UV-B. Methylamine (a weak base) induced H2O2 production and stomatal closure while enhancing the cytosolic alkalization in guard cells under light alone. The rise in cytosolic pH of wild-type guard cells on exposure to UV-B was evident at 15 min and substantial at 45 min while H2O2 production started to largely increase after 60 min. The failure of UV-B-induced H2O2 production in AtrbohD/F guard cells did not affect the changes of guard cell pH during the first 60 min of UV-B radiation, but largely suppressed cytosolic alkalization after 60 min of UV-B radiation. These results indicate that cytosolic alkalization mediates UV-B-induced stomatal closure via activating H2O2 production and that H2O2 production can feedback-enhance cytosolic alkalization in Arabidopsis guard cells.