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BACKGROUND Berberine is a herbicidal chemical that we isolated from Coptis chinensis. In continuation of our program aimed at discovering and developing natural botanical herbicides, we evaluated the herbicidal activities of 39 berberine analogues and developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model. RESULTS Among these 39 analogs, the most active compounds were determined to be worenine chloride and coptisine chloride, with median inhibitory concentration (IC50 ) values on all eight tested weed species of

The qSCN10 locus with broad-spectrum SCN resistance was fine-mapped to a 379-kb region on chromosome 10 in soybean accession PI 567516C. Candidate genes and potential application benefits of this locus were discussed. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most devastating pests of soybean, causing significant yield losses worldwide every year. Genetic resistance has been the major strategy to control this pest. However, the overuse of the same genetic resistance derived primarily from PI 88788 has led to the genetic shifts in nematode populations and resulted in the reduced effectiveness in soybean resistance to SCN. Therefore, novel genetic resistance resources, especially those with broad-spectrum resistance, are needed to develop new resistant cultivars to cope with the genetic shifts in nematode populations. In this study, a quantitative trait locus (QTL) qSCN10 previously identified from a soybean landrace PI 567516C was confirmed to confer resistance to multiple SCN HG Types. This QTL was further fine-mapped to a 379-kb region. There are 51 genes in this region. Four of them are defense-related and were regulated by SCN infection, suggesting their potential role in mediating resistance to SCN. The phylogenetic and haplotype analyses of qSCN10 as well as other information indicate that this locus is different from other reported resistance QTL or genes. There was no yield drag or other unfavorable traits associated with this QTL when near-isogenic lines with and without qSCN10 were tested in a SCN-free field. Therefore, our study not only provides further insight into the genetic basis of soybean resistance to SCN, but also identifies a novel genetic resistance resource for breeding soybean for durable, broad-spectrum resistance to this pest.

The qSCN18 QTL from PI 56756C was confirmed and fine-mapped to improve soybean resistance to the SCN population HG Type 2.5.7 using near-isogenic lines carrying recombination crossovers within the QTL region. The QTL underlying resistance was fine-mapped to a 166-Kbp region on chromosome 18, and the candidate genes were selected based on genomic analyses. Soybean cyst nematode (SCN, Heterodera glycines, Ichinohe) is the most devastating pathogen of soybean. Understanding the genetic basis of SCN resistance is crucial for managing this parasite in the field. Two major loci, rhg1 and Rhg4, were previously characterized as valuable resources for SCN resistance. However, their continuous use has caused shifts in the virulence of SCN populations, which can overcome the resistance conferred by these two major loci. Reduced effectiveness became a major concern in the soybean industry due to continuous use of rhg1 for decades. Thus, it is imperative to identify sources of SCN resistance for durable SCN management. A novel QTL qSCN18 was identified in PI567516C. To fine-map qSCN18 and identify resistance genes, a large backcross population was developed. Nineteen near-isogenic lines (NILs) carrying recombination crossovers within the QTL region were identified. The first phase of fine-mapping narrowed the QTL region to 549-Kbp, whereas the second phase confined the region to 166-Kbp containing 23 genes. Two flanking markers, MK-1 and MK-6, were developed and validated to detect the presence of the qSCN18 resistance allele. Haplotype analysis clustered the fine-mapped qSCN18 region from PI 567516C with the cqSCN-007 locus previously mapped in the wild soybean accession PI 468916. The NILs were developed to further characterize the causal gene(s) harbored in this QTL. This study also confirmed the previously identified qSCN18. The results will facilitate marker-assisted selection (MAS) introducing the qSCN18 locus from PI 567516C into high-yielding soybean cultivars with durable resistance to SCN.

Influenza B virus (IBV) is one of the most important human respiratory viruses: it causes approximately one-third of the global influenza-related disease burden each year. However, compared with the several pathogenicity-related molecular markers that have been identified for influenza A virus (IAV), little is known about potential IBV pathogenicity-related markers. Here, although the IBV strain B/Anhui-Tunxi/1528/2014 (AH1528/14) exhibited a more efficient replication ability in vitro and higher pathogenicity in vivo compared with IBV strain B/Anhui-Baohe/127/2015 (AH127/15), only three amino acids differences (HAA390E, NAN342D and PB1V212I) were observed among their full genomes. The contributions of each amino acid difference to the virus pathogenicity were further investigated. Compared with the wild type IBV virus rAH127, the recombinant virus harbouring a single substitution of HAA390E had a similar phenotype, whereas the recombinant virus harbouring PB1V212I replicated to a moderately higher titre in both MDCK cells and in mice. Notably, the virus harbouring NAN342D showed significantly better growth properties in MDCK cells and higher fatality rates in mice. In addition, the presence of NAN342D dramatically enhanced the viral neuraminidase activity. In conclusion, our study identified a novel IBV molecular marker, NAN342D, that could significantly increase the virulence of IBV in mice.

Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) outside European countries was first detected in Hong Kong Special Administrative Region (Hong Kong, SAR) of China in 2001. Afterwards, EA H1N1 SIVs have become predominant in pig population in this country. However, the epidemiology and genotypic diversity of EA H1N1 SIVs in China are still unknown. Here, we collected the EA H1N1 SIVs sequences from China between 2001 and 2018 and analyzed the epidemic and phylogenic features, and key molecular markers of these EA H1N1 SIVs. Our results showed that EA H1N1 SIVs distributed in nineteen provinces/municipalities of China. After a long-time evolution and transmission, EA H1N1 SIVs were continuously reassorted with other co-circulated influenza viruses, including 2009 pandemic H1N1 (A(H1N1)pdm09), and triple reassortment H1N2 (TR H1N2) influenza viruses, generated 11 genotypes. Genotype 3 and 5, both of which were the reassortments among EA H1N1, A(H1N1)pdm09 and TR H1N2 viruses with different origins of M genes, have become predominant in pig population. Furthermore, key molecular signatures were identified in EA H1N1 SIVs. Our study has drawn a genotypic diversity image of EA H1N1 viruses, and could help to evaluate the potential risk of EA H1N1 for pandemic preparedness and response. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00257-8) contains supplementary material, which is available to authorized users.

Background Tetranychus urticae (T. urticae) Koch is an important pest of vegetable crops worldwide. In this study, bioassays were carried out to analyze the resistance risk, multi-resistance and management of T. urticae Koch to bifenthrin, bifenazate and cyflumetofen on cowpea. Results The resistance ratios of the adult T. urticae population to bifenthrin (G16), bifenazate (G12) and cyflumetofen (G12) were 31.29, 9.38 and 5.81, respectively. Realized heritability (h 2 ) analysis showed that, under a selection pressure of 50-90% mortality, the generations needed to increase 10-fold LC50 values of bifenthrin, bifenazate and cyflumetofen were 3.64-8.05, 5.75-12.71, and 10.93-24.15, respectively. No obvious multi-resistance among these three acaricides was observed. Synergist bioassay results showed that microsomal multifunctional oxidase (MFO) was involved in bifenthrin resistance of T. urticae, with a synergistic ratio of 22.38. However, MFO and GSTs were not the main factors conferring the resistance to bifenazate. MFO, glutathione S-transferases(GSTs), together with esterase contributed to the development of the resistance to cyflumetofen. Additionally, the toxicity selection index test showed that bifenazate was safe to the natural enemy Neoseiulus barkeri (N. barkeri) with a toxicity selection index (TSI) >484.85, while bifenthrin was the least safe (TSI = 0.92). Conclusions These results demonstrated the T. urticae developed higher resistance risk to bifenthrin compared to bifenazate and cyflumetofen and no obvious multi-resistance among these three acaricides, providing guidance for designing appropriate strategies for the effective application of bifenthrin, bifenazate and cyflumetofen in the field and delaying the development of insecticide resistance. © 2019 Society of Chemical Industry.

Licochalcone A (LCA) was found to possess anticancer effects. This study aimed to investigate the anticancer effects and mechanisms of LCA in melanoma. A375 and B16 melanoma cells were stimulated with LCA, MTT assay was used to assess cell proliferation. Expression of miR-142-3p, microphthalmia-associated transcription factor (MITF, which regulates melanin production) and autophagy-related genes was determined by Real-time PCR or western blot. The apoptosis was analyzed by flow cytometry and caspase-3 activity. The roles of miR-142-3p and Ras homolog enriched in brain (Rheb) in LCA-affected cells were investigated by gain- and loss-of functions. LCA inhibited proliferation and MITF expression, but increased apoptosis and autophagy of melanoma cells. Moreover, LCA elevated miR-142-3p expression, but decreased its target gene Rheb expression. The effects of LCA on melanoma cells were abrogated by miR-142-3p inhibitor or Rheb overexpression. LCA suppressed mTOR signaling activation via Rheb. Additionally, rapamycin (a mTOR antagonist) notably attenuated the effects of Rheb on the autophagy, proliferation, apoptosis, and MITF expression in LCA-treated melanoma cells. In conclusion, LCA restrained MITF expression and growth by activating autophagy in melanoma cells via miR-142-3p/Rheb/mTOR pathway. This study suggested that LCA might be a potential therapeutic candidate for prevention and treatment of melanoma.

It has recently been proposed that the Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) is one of the most likely zoonotic viruses to cause the next influenza pandemic. Two main genotypes EA H1N1 viruses have been recognized to be infected humans in China. Our study finds that one of the genotypes JS1-like viruses are avirulent in mice. However, the other are HuN-like viruses and are virulent in mice. The molecular mechanism underlying this difference shows that the NP gene determines the virulence of the EA H1N1 viruses in mice. In addition, a single substitution, Q357K, in the NP protein of the EA H1N1 viruses alters the virulence phenotype. This substitution is a typical human signature marker, which is prevalent in human viruses but rarely detected in avian influenza viruses. The NP-Q357K substitution is readily to be occurred when avian influenza viruses circulate in pigs, and may facilitate their infection of humans and allow viruses also carrying NP-357K to circulate in humans. Our study demonstrates that the substitution Q357K in the NP protein plays a key role in the virulence phenotype of EA H1N1 SIVs, and provides important information for evaluating the pandemic risk of field influenza strains.

Background To investigate the trends and correlation between antibacterial consumption and carbapenem resistance in Gram-negative bacteria from 2012 to 2019 in a tertiary-care teaching hospital in southern China. Methods This retrospective study included data from hospital-wide inpatients collected between January 2012 and December 2019. Data on antibacterial consumption were expressed as defined daily doses (DDDs)/1000 patient-days. Antibacterials were classified according to the Anatomical Therapeutic Chemical (ATC) classification system. The trends in antimicrobial usage and resistance were analyzed by linear regression, while Pearson correlation analysis was used for assessing correlations. Results An increasing trend in the annual consumption of tetracyclines, β-lactam/β-lactamase inhibitor (BL/BLI) combinations, and carbapenems was observed (P Acinetobacter baumannii (A. baumannii) significantly increased (P A. baumannii (P Escherichia coli (E. coli; P A. baumannii to carbapenems was positively correlated with cephalosporin/β-lactamase inhibitor (C/BLI) combinations (P P Burkholderia cepacia (B. cepacia) to carbapenems (P P P Enterobacter cloacae (E. cloacae) to carbapenems. Conclusion These results revealed significant correlations between consumption of antibiotics and carbapenem resistance rates in Gram-negative bacteria. Implementing proper management strategies and reducing the unreasonable use of antibacterial drugs may be an effective measure to reduce the spread of carbapenem-resistant Gram-negative bacteria (CRGN), which should be confirmed by further studies.

Background The gut microbiota is known to play a key role in autoimmune diseases. Objectives To identify and compare the characteristics in the gut microbial composition of patients with alopecia areata (AA) and healthy controls (HCs). Methods In a cross-sectional discovery cohort, we enrolled 33 patients with AA and 35 HCs from the same geographic location in Shanghai, China. The 16S rRNA gene sequencing and bioinformatic analyses were conducted to analyze DNA extracted from the subjects. Results The α-diversity of the AA group demonstrated no statistically significant differences compared with the HC group (P > 0.05). However, the overall gut microbial communities in the AA group were distinct from the HCs (P = 0.0096). We also adopted a random forest model to select three AA-associated OTU biomarkers: OTU1237(Achromobacter), OTU257(Megasphaera), and OTU1784(Lachnospiraceae Incertae Sedis). Conclusion The overall gut microbial composition for AA was distinct from that of HCs. The gut microbial markers we identified may potentially be used for earlier diagnosis and as therapeutic targets.

The purpose of the present study was to determine the biological function and associated regulatory mechanism of small nucleolar RNA host gene 14 (SNHG14) in childhood acute myeloid leukaemia (AML). SNHG14 expression was measured via RT‑qPCR in bone marrow tissues from 57 patients with AML and 57 healthy donors. The clinicopathological features of AML patients with low and high SNHG14 expression were analysed. AML cell viability and apoptosis were assessed using MTT and flow cytometry analyses. The starBase online database, and RNA‑binding protein immunoprecipitation and dual luciferase reporter gene assays were employed to analyse the interactions among SNHG14, microRNA (miR)‑193b‑3p and MCL1 apoptosis regulator BCL2 family member (MCL1). SNHG14 was found to be overexpressed in the bone marrow tissues of patients with AML. The French‑American‑British classification and cytogenetics were significantly different between patients with high and low expression of SNHG14. Silencing SNHG14 decreased AML cell proliferation and facilitated apoptosis. SNHG14 functioned as a sponge for miR‑193b‑3p, and miR‑193b‑3p decreased the viability and accelerated the apoptosis rate of AML cells. In addition, miR‑193b‑3p targeted MCL1. Furthermore, silencing SNHG14 resulted in the sponging of miR‑193b‑3p to regulate cell viability, apoptosis, and MCL1 expression in AML. SNHG14 silencing decreased the viability and promoted apoptosis of AML cells by modulating the miR‑193b‑3p/MCL1 axis.

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Arabidopsis seedling materials Basic Protocol 2: Isolation and enrichment of plasma membrane proteins Support Protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by acyl-biotin exchange method Support Protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate Protocol: Pre-resin digestion and peptide-level enrichment.

BackgroundCurrently, Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) are widely prevalent in pigs in China, with sporadic human cases reported as well. As one of the key molecular makers detected in avian H5N1 and H 7N9 viruses and pandemic H1N1 2009 virus, contributions of T271A in PB2 protein to the EA H1N1 viruses are still unknown. In this study, we investigated the effects of residue 271 in PB2 protein on the viral properties of EA H1N1 viruses.MethodsInfectivity, replication, virulence and pathogenicity of the recombinant viruses containing A or T in position 271 in PB2 protein were studied in cells and mice.ResultsThe results showed that the substitution PB2-T271A increased the viral replication in mammalian and avian cell lines. In addition, the mutation enhanced the viral infectivity, virulence and pathogenicity in mice. The viral titers of lung tissue in the rgHuN271A virus were higher than that of the rgHuN271T at 1, 4, and 7 dpi. The MID50 of the rgHuN271A and rgHuN271T virus were 101.1 TCID50 and 101.9 TCID50, respectively. Besides, the substitution of PB2-T271A enhanced the viral polymerase activity in mammalian cells.ConclusionsThe pathogenicity and replication of EA H1N1 virus containing 271A in PB2 protein were higher than the EA H1N1 virus containing 271T in PB2 protein in vivo and in vitro. Therefore, the PB2-T271A mutation should be continually monitored in influenza viruses circulating in pigs and humans.

MicroRNA‑590 (miR‑590) has been revealed as a tumor suppressor, while low‑density lipoprotein receptor‑related protein 6 (LRP6) is considered to act as a tumor promoter. However, their roles and underlying molecular regulatory mechanisms in esophageal squamous cell carcinoma (ESCC) have yet to be fully elucidated. Therefore, the present study aimed to investigate these mechanisms. The expression levels of miR‑590 and LRP6 in human ESCC samples and cell lines were determined using reverse transcription‑quantitative PCR. Bioinformatics analysis was used to predict the relationship between miR‑590 and LRP6, and luciferase assay was performed to validate the relationship between these factors. Transwell assays were used to determine cell migration and invasion, while western blotting assays were used to detect the protein expression levels of LRP6, E‑cadherin, N‑cadherin and Vimentin. The present study demonstrated that miR‑590 was downregulated and LRP6 was upregulated in ESCC tissues and cell lines. Furthermore, it was found that miR‑590 overexpression and LRP6 knockdown inhibited cell migration, invasion and epithelial‑to‑mesenchymal transition (EMT) in ESCC cell lines. Additional mechanistic studies identified that LRP6 was a target of, and was inhibited by, miR‑590. Collectively, the present findings suggested that miR‑590 inhibited the invasion, migration and EMT of ESCC cells by mediating LRP6.

Background Lung adenocarcinoma (LAC) is composed of lepidic, papillary, mucinous, micropapillary and solid components in its parenchyma. Complex responses to therapeutics result from intratumoral heterogeneity. However, it remains confused that what components in a mixed LAC tumor are responsible to the heterogeneous EGFR mutation and PD-L1 expression. Methods We investigated EGFR status via laser microdissection to capture spatially separated cancer cell subpopulations and digital droplet PCR to determine the abundance of EGFR sensitizing mutation and naïve T790M. Whilst, PD-L1 expression level via tumor proportion score (TPS) was evaluated by Ventana immunohistochemistry using SP263 antibody. PD-L1 expression levels were tiered in =50% groups. Results EGFR mutation harbored in 154 (59%) of 261 LAC patients and more frequently occurred in papillary, lepidic and micropapillary constituents. Higher levels of PD-L1 were found in LACs at stage III and IV (68.3%) versus those at stage I and II (31.7%) (P = 0.04). Solid predominant LACs (41.3%) expressed PD-L1 with TPS > =50%, versus mucinous and lepidic LACs (P =50% were associated with patients’ progression-free survival and longer in the P P EGFR-mutated constituents. Heterogeneous PD-L1 expression occurred in the vicinity of stromal tissues. 58.8, 29.4 and 11.8% in ALK positive LACs (N = 17) were found PD-L1 expression via cutoffs of =50%, respectively (P > 0.05). Conclusion Intratumoral genetic heterogeneity of LACs was demonstrated associated with histological patterns. Heterogeneous PD-L1 expression in higher level usually occurred in solid component both in EGFR mutated and EGFR wild-typed LACs. EGFR mutated LACs heterogeneously had sensitizing and resistant mutation and was accompanied with PD-L1 expression, but discordant among histological constituents. Immune checkpoint inhibitor combined with third generation EGFR tyrosine kinase inhibitor should be more effective to these LACs.

Background Dermal papilla cells (DPCs) play a critical role in the regulation of hair follicle (HF) growth, formation, and cycling. DPCs are thought to regulate HF growth through a paracrine mechanism, in which exosomes may play a critical role. Methods DPC-Exos were cutaneously injected into HFs at different HF cycle stages and the effects were evaluated by histological and immunohistochemical analyses. The effects of DPC-Exos on proliferation, migration, and cell cycle status of outer root sheath cells (ORSCs) were evaluated. After treatment of DPC-Exos, changes in mRNA and protein levels of β-catenin and Sonic hedgehog (Shh) in ORSCs were detected. Results DPC-Exos were approximately 105 nm in diameter and expressed tumor susceptibility gene 101, cluster of differentiation (CD)9, and CD63. Injection of DPC-Exos accelerated the onset of HF anagen and delayed catagen in mice. Immunohistochemical analyses revealed that β-catenin and Shh levels were upregulated in the skin. In vitro, DPC-Exo treatment enhanced ORSC proliferation and migration, and stimulated the expression of β-catenin and Shh. Conclusion DPC-Exos contribute to the regulation of HF growth and development, and provide a potential avenue for the treatment of hair loss.

The Drosophila ovary provides an attractive model for studying the extrinsic or intrinsic factors that regulate the fate of germline stem cells (GSCs). Using this model, we identified a new role for Drosophila spaghetti (spag), encoding a homolog of human RNA polymerase II-associated protein 3 (RPAP3), in regulating the fate of ovarian GSCs. Results from spag knockdown and genetic mosaic studies suggest that spag functions as an intrinsic factor for GSCs maintenance. Loss of Spag by, either spag RNAi or null mutation failed to trigger apoptosis in ovarian GSCs. Overexpression of spag led to negligible increases in the number of GSC/Cystoblast (CB) cells, suggesting that an excess of Spag is not sufficient to accelerate the proliferation of GSCs or delay CBs' differentiation. Our study provides evidence supporting that spag is involved in adult stem cells maintenance. In addition, the RNAi screen results showed that knockdown of Hsp90, one of known Spag interacting partners, led to loss of ovarian GSCs in Drosophila. Heterozygous mutations in hsp90 (hsp90/+) dramatically accelerated the GSC loss in spag RNAi ovaries, suggesting that the Spag-contained complex possibly plays an essential role in controlling the GSCs fate.

BACKGROUND The two-spotted spider mite, Tetranychus urticae Koch is an important pest of cotton. We investigated the efficacy of dimethoate in controlling T. urticae by drip irrigation. Greenhouse and field experiments were carried out to determine the efficacy of dimethoate to T. urticae and the absorption and distribution of dimethoate in cotton. RESULTS Greenhouse results showed that cotton leaves received higher amounts of dimethoate compared with cotton roots and stems, with higher amounts in young leaves compared with old leaves and cotyledon having the lowest amounts among leaves. Field results showed the efficacy of dimethoate to T. urticae by drip irrigation varied by volume of dripping water, soil pH and dimethoate dosage. Dimethoate applied at 3.00 kg ha-1 with 200 m3 ha-1 water at weak acidic soil pH (5.70-6.70) through drip irrigation can obtain satisfactory control efficacy (81.49%, 7 days) to T. urticae, without negatively impacting on its natural enemy Neoseiulus cucumeris. The residue of dimethoate in all cotton seed samples were not detectable. CONCLUSIONS These results demonstrate the effectiveness of applying dimethoate by drip irrigation for control of T. urticae on cotton. This knowledge could aid in the applicability of dimethoate by drip irrigation for field management of T. urticae populations. © 2017 Society of Chemical Industry.

Emerging evidence supports that stem cells are regulated by both intrinsic and extrinsic mechanisms. However, factors that determine the fate of stem cells remain incompletely understood. The Drosophila testis provides an exclusive powerful model in searching for potential important regulatory factors and their underlying mechanisms for controlling the fate of germline stem cells (GSCs). In this study, we have found that Drosophila gilgamesh (gish), which encodes a homologue of human CK1-γ (casein kinase 1-gamma), is required intrinsically for GSC maintenance. Our genetic analyses indicate gish is not required for Dpp/Gbb signaling silencing of bam and is dispensable for Dpp/Gbb signaling-dependent Dad expression. Finally, we show that overexpression of gish fail to dramatically increase the number of GSCs. These findings demonstrate that gish controls the fate of GSCs in Drosophila testis by a novel Dpp/Gbb signaling-independent pathway.

S-metolachlor is a promising alternative to metolachlor. However, the extensive use of S-metolachlor as herbicide in vegetable fields in China has caused concerns about its environmental fate. Here we investigated the effects of temperature, relative humidity (RH) and PH on the degradation rate of S-metolachlor in vegetable soil. The degradation rates of S-metolachlor increased with increasing temperatures and RH and either acidic or basic PH facilitates S-metolachlor degradation. The degradation of S-metolachlor under these conditions followed the first-order kinetics resulting in the half-lives (T-1/2) ranging from 12.18 d to 70.71 d at 5–35°C, and 27.28 d to 53.72 d at RH 30–90%; and 29.62–19.69 d at pH 6–8. Stronger response of soil enzymes including catalase, dehydrogenase, urease and cellulase to S-metolachlor was detected in soil with high organic matter. PLFA profiles showed that, totally, the microbe populations including actinomycetes, fungi and bacteria increased gradually in the first 14 days after the treatment and decreased from 14d to 28d after the treatment. All the S-metolachlor treatments caused the increase of aerobe and anaerobe. High S-metolachlor concentration, 13.9 mg/kg, could cause significant variation at the first 2 weeks, stimulating growth of the entire soil microbial community. These findings might have practical implications for the fate of S-metolachlor residue in vegetable fields. Environmental factors, especially temperature, relative humidity and pH should be considered in combination with the appropriate application dose of S-metolachlor for achieving satisfactory weed-control efficacy, reducing runoff, and minimizing effects on environmental quality.

Main conclusion: Jasmonic acid (JA) negatively regulates stomatal development by promoting LCD expression and hydrogen sulfide (H2S) biosynthesis. H2S inhibits the initiation of stomata formation and acts upstream of SPEECHLESS. Abstract: Stomatal development is strictly regulated by endogenous signals and environmental cues. We recently revealed that jasmonic acid (JA) negatively regulates stomatal development in Arabidopsis thaliana cotyledons (Han et al., Plant Physiol 176:2871-2885, 2018), but the underlying molecular mechanism remains largely unknown. Here, we uncovered a role for H2S in regulating stomatal development. The H2S scavenger hypotaurine reversed the JA-induced repression of stomatal development in the epidermis of wild-type Arabidopsis. The H2S-deficient mutant lcd displayed increased stomatal density and stomatal index values, which were rescued by treatment with sodium hydrosulfide (NaHS; an H2S donor) but not JA, suggesting that JA-mediated repression of stomatal development is dependent on H2S biosynthesis. The high stomatal density of JA-deficient mutants was rescued by exogenous NaHS treatment. Further analysis indicated that JA positively regulates LCD expression, L-cysteine desulfhydrases (L-CDes) activity, and endogenous H2S content. Furthermore, H2S represses the expression of stomate-associated genes and functions downstream of stomate-related signaling pathway components TOO MANY MOUTHS (TMM) and STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) and upstream of SPEECHLESS (SPCH). Therefore, H2S acts downstream of JA signaling to regulate stomatal development in Arabidopsis cotyledons.

Therapeutic leukaemia vaccines have shown modest potency. Here, we show that the co-encapsulation of a leukaemia-associated epitope peptide highly expressed in leukaemia patients and of the immune checkpoint inhibitor anti-programmed-cell-death-protein-1 (anti-PD-1) in degradable poly(lactic acid) microcapsules resulted in the sustained release of the peptide and of the antibody, which led to the recruitment of activated antigen-presenting cells to the injection site, their uptake of the peptide and the transportation of the anti-PD-1 antibody to lymph nodes, enhancing the expansion of epitope-specific T cells and the activation of cytotoxic T cells. After single subcutaneous injections of vaccine formulations with different epitope peptides, mice bearing leukaemia xenografts derived from humanized cell lines or from primary cells from patients showed better therapeutic outcomes than mice receiving repeated injections of free antigen, antibody and a commercial adjuvant. The sustained release of a tumour-associated peptide and of anti-PD-1 may represent a generalizable strategy for boosting antitumour immune responses to leukaemia. The sustained release of a leukaemia-associated epitope peptide and of anti-PD-1 antibody co-encapsulated in degradable microcapsules results in superior therapeutic outcomes in mouse models of leukaemia.

A new bacterial disease of citrus was recently identified in Florida and is here named bacterial brown leaf spot (BBLS) of citrus. BBLS-infected citrus leaves from the field displayed circular, brownish, flat lesions with slightly raised and water-soaked margins surrounded by a chlorotic halo. Based on Biolog carbon source metabolic “fingerprinting”, fatty acid analysis, and sequence analysis of partial 16S rDNA, gyrB, and rpoD genes, the causal agent of the disease was identified as Burkholderia andropogonis. Pathogenicity of these B. andropogonis isolates taken from multiple citrus leaves with BBLS was tested by various inoculation methods on three species of citrus as well as on carnation, corn, and sorghum. All isolates infected carnation, corn, and sorghum with varying degrees of pathogenicity. Variation among citrus isolates in pathogenicity was also observed in high titer (108 CFU/ml) inoculations of citrus leaves, ranging from a hypersensitive-like response to canker-like lesions. When the inoculum concentration was low (106 CFU/ml), only necrotic spots or small lesions slowly developed with all strains. Growth of B. andropogonis in citrus was relatively slow, tissue wounding appeared necessary for symptom appearance with many isolates, and field samples were recovered only after severe storms, indicating that this wide-host-range bacterium is a weak, opportunistic pathogen of citrus.

Soybean (Glycine max L.) is an economically important crop providing a great source for vegetable oil and protein. Yield losses of soybean under current climate change keep increasing, despite the progressive increase in yield through breeding and management practices since the 1960s. Conventional breeding facilitated the development of high-quality soybeans with enhanced tolerance to severe environmental fluctuations such as drought, flooding, heat, and salinity. However, conventional approaches are laborious, time consuming, and looks inefficient to fulfill the increasing demands of the growing world population. The advances in marker-assisted and genomics-assisted breeding, sequencing technologies, and bioinformatics tools have enabled the soybean improvement at a faster pace. The rapidly accumulating genomic resources have enabled the development of molecular markers associated with many important quantitative trait loci, provided a clear picture of genomic variations in soybean germplasm, and identified key genes for genetic engineering. This knowledge is being utilized to facilitate the development of climate-smart soybeans. In this chapter, we discuss and summarize the advances in soybean improvement through conventional and genomics-assisted breeding, genetic engineering approaches, and available bioinformatics tools for soybean. This chapter also highlights soybean genetic resources, diversity analysis, association mapping, as well as recent strategies such as gene editing and nanotechnology application in soybean breeding programs. This information could facilitate the incorporation of climatic-smart traits in breeding for more stable soybean production with the changing climate.

Human skin organ culture (hSOC) is a simple but highly instructive and clinically relevant skin research method. It has been used for decades to study the development, differentiation, and function as well as the response to wounding or test agents of intact human skin in the presence of its appendages and all resident cell populations. hSOC has also proven useful in toxicological and oncological studies and studies of skin aging (both chronological aging and photoaging), skin energy metabolism, skin immunology, pigmentation biology, and cutaneous (neuro-)endocrinology and neurobiology. The pathobiology and treatment of various dermatoses can also be assessed ex vivo by organ-culturing intact lesional human skin. In addition to morphological analyses by routine histochemistry, quantitative (immuno)histomorphometry has proven to be an excellent tool for quantitating and localizing protein expression patterns in defined skin compartments and distinct cell populations using a relatively small amount of precious human tissue. Finally, more recent technological advances, such as siRNA-mediated gene silencing and sensory reinnervation of hSOCs, have further extended the range of methodological applications for the ex vivo study of human skin; it has emerged as the ultimate preclinical assay system for investigative dermatology, including the testing of drugs, cosmeceuticals and nutraceuticals and more, and is just one step below human skin xenotransplant in vivo mouse models and clinical trials. Here, we critically review the renaissance and variety of hSOC assays, their applications and limitations, and we critically compare them with 3D skin "equivalent" assays. The review closes with perspectives on how this ancient but highly informative and physiologically relevant ex vivo skin research method may be further developed in the future.

DECORIN is a prototypical member of the small leucine-rich proteoglycan (SLRP) family that plays important roles in numerous biological processes and cellular biological pathways. We previously showed that Decorin expression was highly enhanced in mouse dorsal hair follicles (HFs) during the anagen phase and was reduced during the catagen and telogen phases, suggesting that Decorin might modulate follicular cycling and morphogenesis. In this study, to further clarify the effects of DECORIN on hair cells and the cycling transition, an in vitro overexpression strategy and Decorin-null (Dcn-/- ) mice were used to investigate the effects of DECORIN on outer root sheath (ORS) keratinocytes. DECORIN overexpression significantly enhanced proliferation and migration in ORS keratinocytes in vitro. Moreover, DECORIN overexpression upregulated the mRNA and protein expression levels of WNT10b, β-CATENIN and LEF1. The DECORIN overexpression-induced increase in the proliferation and migration of ORS keratinocytes was partially inhibited by a Wnt/β-catenin inhibitor. Furthermore, Dcn-/- mice had a shortened anagen phase and lower levels of β-catenin expression than were observed in wild-type mice in imaging and histological analyses. Taken together, these findings suggest that DECORIN promotes the proliferation and migration of ORS keratinocytes in vitro and maintains hair anagen in mice.

It is well known that cyclin B3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, we show that Drosophila cycB3 plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans.

Although primary mucin-producing adenocarcinoma of the lung is uncommon, each subtype has distinct clinical, pathological, molecular, and prognostic characteristics. This study aimed to determine correlations between clinical and pathological features and genetic phenotypes with the prognosis. We immunohistochemically examined the protein levels of thyroid transcription factor 1 (TTF-1), Napsin A, and anaplastic lymphoma kinase (ALK) and genetically examined epidermal growth factor receptor (EGFR) and KRAS mutations in these mucin-producing tumors. A total of 75 cases of mucin-producing adenocarcinoma of the lung were examined. ALK protein positivity was 33.3 % (25/75), and primarily occurred in solid predominant with mucin production subtype (SA). KRAS mutations occurred in 22.7 % (17/75) of patients, predominantly in invasive mucinous adenocarcinoma (IMA). Positive TTF-1 and Napsin A expression was more common in SA, while they were both negative in IMA. The 1-, 3-, and 5-year progression-free survival rates of mucin-producing lung adenocarcinoma were 85, 64, and 38 %, respectively; the overall survival rates were 90, 67, and 50 %, respectively. Larger tumors, advanced stage, and lymph node metastasis were associated with poor prognosis. Mucinous minimally invasive adenocarcinoma (m-MIA) had the best prognosis, followed by IMA, SA, and acinar or papillary predominant adenocarcinoma with mucin production (A/P). KRAS mutations were an independent positive prognostic factor for postoperative progress.

Background c-ros oncogene 1 (ROS1) rearrangement presents one of the newest molecular targets in non-small cell lung cancer (NSCLC). ROS1 rearrangement is predominantly found in adenocarcinoma cases and is exclusive to other oncogenes, such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK). The aim of this study was to investigate the clinicopathological characteristics and outcomes of ROS1-rearranged patients with lung adenocarcinoma without EGFR and KRAS mutations and ALK rearrangements. Methods Wild-type EGFR/KRAS/ALK patients with lung adenocarcinoma were selected from Beijing Chest Hospital. Specimens were conducted in tissue microarrays. ROS1 rearrangement was screened using fluorescence in situ hybridization. Results Our study included 127 patients with lung adenocarcinoma without EGFR and KRAS mutations and ALK rearrangements. ROS1 rearrangement was detected in five (3.9%) of the 127 patients. Compared with ROS1-negative patients, the positive rate of ROS1 in female patients was significantly higher than in male patients (9.8% vs. 0.0%, P = 0.009). There were no differences in age, smoking status, stage or histological subtype between ROS1-positive and ROS1-negative patients. No significant difference in survival was detected between the ROS1-positive and ROS1-negative patients. Conclusions ROS1 rearrangement is a rare subset of lung adenocarcinoma. In 127 patients with lung adenocarcinoma, 3.9% of ROS1-positive patients with wild-type EGFR/KRAS/ALK were found.

Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM) may lead to preeclampsia (PE). We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO) annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8) related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE).

Primary pulmonary mucinous adenocarcinoma (PPMA) is one of the important subtypes of lung adenocarcinoma. Detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements and of KRAS and epidermal growth factor receptor (EGFR) mutations will help in diagnosing and predicting treatment outcome. The aim of this study was to investigate the clinicopathological significance of ALK rearrangements, KRAS and EGFR mutations in PPMA. ALK expression was detected immunohistochemically. KRAS and EGFR mutations were determined by the amplification refractory mutation system. Seventy-three patients of PPMA were enrolled. ALK rearrangements were detected in 34.2 % of patients and were more frequent in upper/middle lobe, stage III-IV, lymphatic permeation-positive patients and non-smokers. ALK rearrangements were significantly increased in the solid tumor predominant with mucin production subtype, and in special tissue structures, including signet ring cells, cribriform, and micropapillary patterns. KRAS mutations were observed in 23.3 % of patients and were more prevalent in invasive mucinous adenocarcinoma and lower lobe tumors. Only one case of ALK rearrangements harbored KRAS mutation, and no cases manifested with the coexistence of ALK rearrangements and EGFR mutations. KRAS and EGFR co-mutation was detected in one case. PPMA patients with ALK rearrangements or KRAS mutation represent a unique subtype in NSCLC. The results provide basis data for target therapy screening of PPMA patients.

This study was designed to investigate the protective effects of Acanthus ilicifolius alkaloid A [4-hydroxy-2-benzoxazolone (HBOA)] and its acetylated derivatives including 4-acetoxy-2-benzoxazolone (TC-2) and 3-acetyl-4-acetoxy-2-benzoxazolone (TC-3) on carbon tetrachloride (CCl4 )-induced liver fibrosis in rats. Sprague-Dawley rats were given CCl4 twice per week for 8 weeks to induce liver fibrosis. Then, they were treated with HBOA, TC-2, and TC-3 daily for 4 weeks, respectively. The serum indicators including total protein (TP), albumin (Alb), globulin, hyaluronic acid (HA), and laminin (LN) were measured by commercial kits. The messenger ribonucleic acid expression of adiponectin, peroxisome proliferator-activated receptor-γ (PPAR-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1 ) and Toll-like receptor 4 (TLR4 ) was determined by reverse-transcriptase -PCR. The proteins of adiponectin, TGF-β1 , α-smooth muscle actin (α-SMA), and TLR4 were also detected by the immunohistochemical assay. The results showed that HBOA, TC-2, and TC-3 significantly attenuated the fibrotic degree induced by CCl4 as evidenced by higher levels of TP, Alb, adiponectin, and PPAR-γ, which in turn decreased the proliferation of hepatic stellate cells. Moreover, those drugs markedly decreased the levels of HA, LN, TNF-α, IL-6, TGF-β1 , α-SMA, and TLR4 . Our study indicates that HBOA, TC-2, and TC-3 have beneficial effects against liver fibrosis, and the mechanisms may be related to the inhibition of inflammatory response.

Climate change has increased the occurrence of extreme weather patterns globally, causing significant reductions in crop production, and hence threatening food security. In order to meet the food demand of the growing world population, a faster rate of genetic gains leading to productivity enhancement for major crops is required. Grain legumes are an essential commodity in optimal human diets and animal feed because of their unique nutritional composition. Currently, limited water is a major constraint in grain legume production. Root system architecture (RSA) is an important developmental and agronomic trait, which plays vital roles in plant adaptation and productivity under water-limited environments. A deep and proliferative root system helps extract sufficient water and nutrients under these stress conditions. The integrated genetics and genomics approach to dissect molecular processes from genome to phenome is key to achieve increased water capture and use efficiency through developing better root systems. Success in crop improvement under drought depends on discovery and utilization of genetic variations existing in the germplasm. In this review, we summarize current progress in the genetic diversity in major legume crops, quantitative trait loci (QTLs) associated with RSA, and the importance and applications of recent discoveries associated with the beneficial root traits towards better RSA for enhanced drought tolerance and yield.

Background Drought (Water deficit, WD) poses a serious threat to extensively economic losses of trees throughout the world. Chinese dwarf cherry (Cerasus humilis) is a good perennial plant for studying the physiological and sophisticated molecular network under WD. The aim of this study is to identify the effect of WD on C. humilis through physiological and global proteomics analysis and improve understanding of the WD resistance of plants. Methods Currently, physiological parameters were applied to investigate C. humilis response to WD. Moreover, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in C. humilis leaves subjected to WD (24 d). Furthermore, we also examined the correlation between protein and transcript levels. Results Several physiological parameters, including relative water content and Pn were reduced by WD. In addition, the malondialdehyde (MDA), relative electrolyte leakage (REL), total soluble sugar, and proline were increased in WD-treated C. humilis. Comparative proteomic analysis revealed 46 protein spots (representing 43 unique proteins) differentially expressed in C. humilis leaves under WD. These proteins were mainly involved in photosynthesis, ROS scavenging, carbohydrate metabolism, transcription, protein synthesis, protein processing, and nitrogen and amino acid metabolisms, respectively. Conclusions WD promoted the CO2 assimilation by increase light reaction and Calvin cycle, leading to the reprogramming of carbon metabolism. Moreover, the accumulation of osmolytes (i.e., proline and total soluble sugar) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione s-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under WD. Importantly, the regulation role of carbohydrate metabolisms (e. g. glycolysis, pentose phosphate pathways, and TCA) was enhanced. These findings provide key candidate proteins for genetic improvement of perennial plants metabolism under WD. Electronic supplementary material The online version of this article (doi:10.1186/s12953-017-0117-1) contains supplementary material, which is available to authorized users.

Background: STIM/ORAI-mediated store-operated Ca2+ entry (SOCE) mediates a myriad of Ca2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-offunction mutations in STIM1/ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Objective and Methods: Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Results: Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of STIM1 by a more rigid dimerized TM domain of glycophorin A abolished STIM1 activation. But this adverse effect could be partially reversed by disrupting the TM dimerization interface. Moreover, our study revealed regions that are important for the optimal assembly of hetero-oligomers composed of full-length STIM1 with its minimal STIM1-ORAI activating region, SOAR. Conclusions: Our study clarifies the roles of major STIM1 functional domains in maintaining a quiescent configuration of STIM1 to prevent preactivation of SOCE.

Purpose: To study the effect of Compound Bieshe Kang’ai (CBK) on proliferation and apoptosis in colorectal cancer cells.Methods: HCT116 colorectal cancer cells and FHs 74 Int intestinal cells were treated with CBK, followed by determination of cell proliferation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Caspase-9 and caspase-3 activities as well as protein expressions of Bcl-2 and BAX, and mRNA levels of caspase-9, caspase-3, Bcl-2 and BAX in HCT116 cells were evaluated, followed by examination of the morphological alterations of HCT116 cells with Hoechst 33342 staining.Results: CBK suppressed proliferation of HCT116 cells in a concentration- and time-dependent pattern, without cytotoxicity to FHs 74 Int cells. CBK also elevated caspase-9 and caspase-3 activities, mitigated protein translation of Bcl-2 and augmented that of BAX. It also enhanced mRNA transcriptions of caspase-9, caspase-3 and BAX, but decreased that of Bcl-2 in HCT116 cells in a concentrationdependent manner, as well as induced cancer cell shrinkage, nuclear fragmentation and chromatin condensation.Conclusion: The findings highlight CBK as a promising therapeutic agent for colorectal cancers, by retarding proliferation and inducing apoptosis in cancer cells.Keywords: Apoptosis, BAX, Bcl-2, Cancer, Caspase, Compound Bieshe Kang’ai, Chromatin condensation, Nuclear fragmentation

Background/Aims: Previous studies have indicated that long non-coding RNAs (lncRNA) are related to the occurrence and development of many human diseases, such as cancer and the HELLP and the brachydactyly syndromes. However, studies of LncRNA in heart failure have not yet been reported. Here, we investigated cardiac lncRNA expression profiles in the myocardial-specific knockout pdk1 gene (KO) mouse model of heart failure. Methods: Cardiac samples were obtained from PDK1 KO and WT mice on postnatal (P) day 8 (P8) and day 40 (P40), and lncRNA expression profiles were analyzed by sequencing and screening using the Arraystar mouse lncRNA microarray. Quantitative real-time PCR analysis of these lncRNAs confirmed the identity of some genes. Results: Comparisons of the KO and control groups showed fold changes of >1.5 in the expression levels of 2,024 lncRNAs at P8, while fold changes of >2 in the expression levels of 4,095 lncRNAs were detected at P40. Nineteen lncRNAs were validated by RT-PCR. Bioinformatic and pathway analyses indicated that mkk7, a sense overlap lncRNA, may be involved in the pathological processes of heart failure through the MAPK signaling pathway. Conclusion: These data reveal differentially expressed lncRNA in mice with a myocardial-specific deletion of the pdk1 gene, which may provide new insights into the mechanism of heart failure in PDK1 knockout mice.

Acanthus ilicifolius alkaloid A (4-hydroxy-2(3H)benzoxazolone, HBOA) is a naturally occurring compound that has been separated from Acanthus ilicifolius. Previous studies have reported the beneficial effects of HBOA on HSC-T6 cells. This study was undertaken in order to synthesize HBOA and two of its derivatives, specifically, 4-acetoxy-2(3H)-benzoxazolone (AcO-BOA) and 3-acetyl-4-acetoxy-2-benzoxazolone (TC-3), and to investigate the hepatoprotective potentials of these three compounds on CCl4-induced liver injury in mice. HBOA was prepared from 2-nitroresorcinol by a 'one pot' reduction and subsequent cyclization with urea. The acyl derivatives, AcO-BOA and TC-3, were prepared from HBOA using a substitution reaction. The compounds were synthesized with good yields (63.08–68.22%). An acute liver injury model was established by administering CCl4 intraperitoneally to Kunming mice. The mice were then intragastrically administered bifendate (150 mg/kg) or the synthesized compounds at three different doses (200, 100 and 50 mg/kg). The treatment with CCl4 was observed to increase the levels of aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH) and malondialdehyde (MDA) and decrease the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (Gpx) in the liver tissues of the mice. Furthermore, treatment with CCl4 elevated the expression level of the proinflammatory mediator TNF-α. However, HBOA and its derivatives attenuated the changes induced by CCl4. Furthermore, CCl4-induced histopathological changes were reduced by treatment with these compounds. These results suggest that HBOA and its acyl derivatives are able to significantly alleviate the hepatotoxicity induced by CCl4 in mice.

Fatty acid-binding protein 3 (FABP3) is a low molecular weight protein with distinct tissue distribution, which may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. We have previously shown FABP3 was more highly expressed in myocardium with ventricular septal defects than in normal myocardium and furthermore, that overexpression of FABP3 causes mitochondrial dysfunction and induces apoptosis in the P19 mouse teratocarcinoma cell line (P19), which is a suitable model for the investigation of cardiac differentiation at the molecular and functional levels. α-Lipoic acid (α-LA), a natural dithiol compound with antioxidant properties, has been reported to protect mitochondrial function in cells. In this study, we established an FABP3-overexpressing P19 cell line for the investigation of the impact of α-LA on mitochondrial impairment and apoptosis in these cells. Mitochondrial morphology was evaluated by transmission electron microscopy, while the effects of α-LA on reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), intracellular ATP content and the amount of mitochondrial DNA were analyzed by flow cytometry, a commercially available assay and quantitative real-time PCR, respectively. The results revealed that α-LA ameliorated mitochondrial deformation and decreased intracellular ROS production. Furthermore, the MMP, intracellular ATP synthesis and the amount of mitochondrial DNA were also increased. Most significantly, α-LA was shown to reverse apoptosis. Collectively, our results indicate that abnormalities in FABP3 expression contribute to mitochondrial dysfunction and apoptosis, and that α-LA represents a suitable candidate for development as a treatment for apoptosis-related congenital cardiac malformations.

Huanglongbing (HLB) is currently the most destructive disease of citrus worldwide. Although there is no immune cultivar, field tolerance to HLB within citrus and citrus relatives has been observed at the USDA Picos farm at Ft. Pierce, Florida, where plants have been exposed to a very high level of HLB pressure since 2006. In this study, we used RNA-Seq to evaluate expression differences between two closely related cultivars after HLB infection: HLB-tolerant "Jackson" grapefruit-like-hybrid trees and HLB susceptible "Marsh" grapefruit trees. A total of 686 genes were differentially expressed (DE) between the two cultivars. Among them, 247 genes were up-expressed and 439 were down-expressed in tolerant citrus trees. We also identified a total of 619 genes with significant differential expression of alternative splicing isoforms between HLB tolerant and HLB susceptible citrus trees. We analyzed the functional categories of DE genes using two methods, and revealed that multiple pathways have been suppressed or activated in the HLB tolerant citrus trees, which lead to the activation of the basal resistance or immunity of citrus plants. We have experimentally verified the expressions of 14 up-expressed genes and 19 down-expressed genes on HLB-tolerant "Jackson" trees and HLB-susceptible "Marsh" trees using real time PCR. The results showed that the expression of most genes were in agreement with the RNA-Seq results. This study provided new insights into HLB-tolerance and useful guidance for breeding HLB-tolerant citrus in the future.

The micropapillary (MP) subtype has recently been established to be a distinct marker of poor prognosis in lung adenocarcinomas (LACs). According to the 2015 WHO classification system, LAC constituents are required to be precisely reported. T790M mutation and an insertion in exon 20 (E20ins) are associated with EGFR-TKI resistance. A total of 211 LAC patients were involved in this study, and EGFR mutations were determined using an amplification refractory mutation system (ARMS). Sex, smoking history, lymph node status, and clinical stage differed significantly between the EGFR wild type and mutant groups (p EGFR mutation occurred more frequently in female, non-smokers, ACs with papillary (85.7%) or MP components (91.4%) (p EGFR status as the surrounding T790M-mutated MP components. The MP and glomerulus-like portions in AC tumours exhibited a congenial EGFR status, but the acinar cells with papillary cells were heterogeneous. The naïve T790M mutants, although minor in the MP component, dramatically increased after EGFR-TKI therapy and indicate that the MP components feature intrinsic heterogeneity.

Auxin transport and signaling enforce stomatal patterning as well as the size of their stem cell compartment in Arabidopsis. But it remains unknown whether this regulation is also existed in other species. We use Nicotiana as an example to analysis the function of auxin transport inhibitor NAP and BFA on its stomatal developement on the epidermis of cotyledon taking Arabidopsis as control. Here we report that there is an inter-specific difference between Nicotiana and Arabidopsis under exogerous auxin inhibitors (NPA and BFA) treatment in stomatal development. No stomatal development defect can be detected in Nicotiana treated with NPA and BFA under the same concentration which can induce patterning and morphogenesis abnormality of stoma in Arabidopsis.

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca2+-modulated activities with tailored function. DOI: http://dx.doi.org/10.7554/eLife.10024.001, eLife digest Optogenetics is a technique that has been used to study nerve cells for several years. It involves genetically engineering these cells to produce proteins from light-sensitive bacteria, and results in nerve cells that will either send, or stop sending, nerve impulses when they are exposed to a particular color of light. Neuroscientists have learned a lot about brain circuits using the technique, and now researchers in many other fields are giving it a try. There are, however, several challenges to using optogenetics in other types of cells. Nerve cells create a tiny electrical impulses when they are activated, which helps them quickly transmit messages. But other types of cells use more diverse means to communicate and transmit signals. This means that optogenetics techniques must be adapted. Additionally, many cells are located deep in the body and so getting the light to them can be difficult. He, Zhang et al. have now developed an optogenetic system (termed “Opto-CRAC”) that can control immune cells buried deep in tissue. The action of immune cells can be tuned by controlling the flow of calcium ions through gate-like proteins in their membranes. He, Zhang et al. genetically engineered immune cells so that a calcium gate-controlling protein became light sensitive. When the cells were exposed to a blue light the calcium ion gates opened. When the light was turned off, the gates closed. More intense light caused more calcium to enter into the cells. Further experiments then revealed that exposing these engineered immune cells to blue light in the laboratory could trigger an immune response. The next obstacle was getting light to immune cells in a live animal. So, He, Zhang et al. used specific nanoparticles that have been shown to help transmit light deep within tissue. In these experiments, mice were injected with the light-sensitive immune cells and the nanoparticles. Then, a near-infrared laser beam that can transmit into the tissues was pointed at the mice. This caused calcium channels to open in the engineered cells deep in the mice. Finally, further experiments were used to show that this light-based stimulation could boost an immune response to aid the killing of cancer cells. Other scientists will likely use the technique to help them study immune, heart, and other types of cells that use calcium to communicate. DOI: http://dx.doi.org/10.7554/eLife.10024.002

Zhang, M. Q., Powell, C. A., Zhou, L. J., He, Z. L., Stover, E., and Duan, Y. P. 2011. Chemical compounds effective against the citrus Huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ in planta. Phytopathology 101:1097-1103. Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is threatening the survival of the Floridian citrus industry. Currently, there is no established cure for this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic compounds were screened using a propagation test system with ‘Candidatus Liberibacter asiaticus’-infected periwinkle and citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing the ‘Ca. L. asiaticus’ bacterium and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, ‘Ca. L. asiaticus’infected periwinkle cuttings achieved 70% of regeneration rates versus

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

A new iridoid, torrilliolide (1), and a new pyridine alkaloid, torricelline (2), were isolated from the stem barks of Torricellia tiliifolia. Their structures were determined by analysis of their spectroscopic data. Both compounds exhibited cytotoxicity against Spodoptera litura cell line.