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The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

Supplementary Figure from Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia

Supplementary Table from Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia

Background Many studies have elucidated the regulatory roles of long non-coding RNAs in cardiovascular diseases. The current research was conducted to investigate the involvement of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) with microRNA-135a-5p (miR-135a-5p) and hypoxia-inducible factor 1-alpha subunit inhibitor (HIF1AN) in myocardial ischemia-reperfusion (I/R) injury. Methods A hypoxia-reoxygenation (H/R) model was established in rat cardiomyocytes. RNA or protein level analysis was implemented via qRT-PCR and Western blot. MTT assay was conducted for cell viability detection. H/R injury was evaluated using lactate dehydrogenase assay. The assessment of apoptosis was carried out through flow cytometry and caspase-3 assay. Enzyme-linked immunosorbent assay was applied for determining inflammatory cytokines. Target relation analysis was implemented through dual-luciferase reporter, RNA immunoprecipitation and pull-down assays. MALAT1 function in vivo was evaluated using rat H/R model and infarct areas were determined by triphenyltetrazolium chloride staining. Results MALAT1 and HIF1AN were up-regulated in H/R model of cardiomyocytes. H/R promoted cell apoptosis and inflammatory cytokine release in cardiomyocytes, while these effects were ameliorated by MALAT1 or HIF1AN knockdown. HIF1AN up-regulation reverted the protective function by MALAT1 knockdown in H/R cardiomyocytes. MALAT1 upregulated the HIF1AN expression through absorbing miR-135a-5p. Silencing MALAT1 activated the PI3K/AKT pathway via downregulating HIF1AN. MALAT1 inhibition reduced infarct and apoptosis by depending on the miR-135a-5p/HIF1AN axis in vivo. Conclusion MALAT1 aggravate the H/R-stimulated myocardial I/R injury through the mediation of miR-135a-5p/HIF1AN axis.

Purpose:PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki.Experimental Design:Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.Results:pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively.Conclusions:Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

PurposePhosphatidylinositol 3-kinase inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response, development of treatment intolerance or resistance remains a clinically challenging. Prediction of individual treatment responses based on clinically actionable biomarkers is needed to overcome these challenges. Here, we investigated whetherex vivofunctional responses to targeted therapies can stratify responders to idelalisib and guide precision medicine in CLL.Experimental designCLL cells from treatment naïve, idelalisib-responding, and idelalisib-refractory/intolerant patients (n=33 in total) were profiled against ten PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.ResultsAmong the ten PI3Ki studied, pan-PI3Ki were most effective at inhibiting PI3K signaling and cell viability, and they showed activity also in CLL cells from idelalisib-refractory/intolerant patients. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+and CD8+T cells in addition to CD19+B cells, while it did not significantly affect T cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Based onex vivodrug sensitivity testing, a relapsed CLL patient was treated with idelalisib plus venetoclax, and the patient achieved a partial response. A more systematic analysis revealed that CLL cells from patients with a long-term response to idelalisib showed significantly higher drug sensitivities to 73 drug combinations at baseline compared to short-term responders.ConclusionsOur findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and demonstrate thatex vivofunctional profiling may guide precision medicine and predict treatment responses of individual CLL patients.TRANSLATIONAL RELEVANCEThe phosphatidylinositol 3-kinase inhibitors (PI3Ki) idelalisib and duvelisib are approved for relapsed chronic lymphocytic leukemia (CLL), but their use has been limited by severe toxicity and acquired resistance. Identification of biomarkers that predict individual treatment responses, as well as alternative treatment vulnerabilities in PI3Ki refractory/intolerant patients, is needed to optimally tailor CLL therapy. We performed functional analyses of CLL cells from treatment naïve, idelalisib-responding and idelalisib-refractory/intolerant patients to identify clinically actionable biomarkers. We show that CLL cells from idelalisib-refractory/intolerant patients remain sensitive to pan-PI3Ki and PI3Ki plus venetoclax combinations.Ex vivodrug sensitivity testing was used to guide treatment of a relapsed CLL patient who obtained a partial response after idelalisib plus venetoclax therapy. A systematic analysis of drug sensitivities to 73 drug combinations stratified responders to idelalisib using baseline samples from short-term and long-term responders to idelalisib. Our study demonstrates the power of functional precision medicine in relapsed CLL.

A large number of long non-coding RNAs have been confirmed to play vital roles in regulating various biological processes. Abnormal expression of growth arrest-specific transcript 5 (GAS5) is reported to be involved in the development of atherosclerosis (AS). This work is to explore the detailed mechanism underling how GAS5 regulates AS progression. We found that the abundance of GAS5 was markedly increased, and miR-135a was decreased in AS patient serums and ox-LDL-induced human THP-1 cells dose and time dependently. Interference of GAS5 suppressed inflammation and oxidative stress induced by ox-LDL in THP-1 cells. Mechanistically, GAS5 acted as a molecular sponge of microRNA-135a (miR-135a). Rescue assays indicated that knockdown of miR-135a partially rescued small interference RNA for GAS5-inhibited inflammatory cytokines release and oxidative stress in ox-LDL-triggered THP-1 cells. In conclusion, the absence of GAS5-inhibited inflammatory response and oxidative stress induced by ox-LDL in THP-1 cells via sponging miR-135a, providing a deep insight into the molecular target for AS treatment.

Purpose:PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki.Experimental Design:Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.Results:pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively.Conclusions:Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

Many studies have elucidated the regulatory roles of long non-coding RNAs in cardiovascular diseases. The current research was conducted to investigate the involvement of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) with microRNA-135a-5p (miR-135a-5p) and hypoxia-inducible factor 1-alpha subunit inhibitor (HIF1AN) in myocardial ischemia-reperfusion (I/R) injury.A hypoxia-reoxygenation (H/R) model was established in rat cardiomyocytes. RNA or protein level analysis was implemented via qRT-PCR and Western blot. MTT assay was conducted for cell viability detection. H/R injury was evaluated using lactate dehydrogenase assay. The assessment of apoptosis was carried out through flow cytometry and caspase-3 assay. Enzyme-linked immunosorbent assay was applied for determining inflammatory cytokines. Target relation analysis was implemented through dual-luciferase reporter, RNA immunoprecipitation and pull-down assays. MALAT1 function in vivo was evaluated using rat H/R model and infarct areas were determined by triphenyltetrazolium chloride staining.MALAT1 and HIF1AN were up-regulated in H/R model of cardiomyocytes. H/R promoted cell apoptosis and inflammatory cytokine release in cardiomyocytes, while these effects were ameliorated by MALAT1 or HIF1AN knockdown. HIF1AN up-regulation reverted the protective function by MALAT1 knockdown in H/R cardiomyocytes. MALAT1 upregulated the HIF1AN expression through absorbing miR-135a-5p. Silencing MALAT1 activated the PI3K/AKT pathway via downregulating HIF1AN. MALAT1 inhibition reduced infarct and apoptosis by depending on the miR-135a-5p/HIF1AN axis in vivo.MALAT1 aggravate the H/R-stimulated myocardial I/R injury through the mediation of miR-135a-5p/HIF1AN axis.

Immunoassay in biomedical field has attracted more and more attention, and surface plasmon technology is an effective detection method. Electroluminescent devices are suitable for excitation light source of surface plasma technology because of its low energy consumption and small size. In this paper, a light-emitting chip based on MIS structure is designed, and its electroluminescence principle and its internal electron tunneling principle are studied. Simultaneously, the structure and production process of the chip are proposed. The plasma oscillation frequency of doped silicon and the asymptotic frequency corresponding to the SPP dispersion curve of each interface in each MIS junction are calculated and analyzed, and the relationship between the asymptotic frequency and the wavelength of the light emission spectrum of the device is deduced. Finally, the luminescence spectrum wavelength corresponding to the asymptotic frequency of each interface is obtained, which lays a theoretical foundation for future experiments to verify whether the asymptotic frequency of each interface of the MIS junction is consistent with the peak value of the device luminescence spectrum.

In recent years, the plasma gap resonance maintained by metal-film-coupled nanostructures has attracted extensive attention. This mainly originates from its flexible control of the spectral response and significantly enhanced field strength at the nanoparticle–film junction. In the present study, the tunability of local surface plasmon resonances (LSPRs) of nanorods coupled to a gold film is studied theoretically. To this end, the plasmonic resonances in the nanostructure of individual silver nanorod–gold film (AgNR-film) with different parameters are investigated. Obtained results show that the refractive index sensitivity (S) of nanostructures to the environment increases as the aspect ratio (Ar) of nanostructures increase. It is found that when the aspect ratio (Ar) is set to 3.5, the figure of merit (FOM) is the highest. Moreover, the variation in the gap distances of the nanorod monomer–gold film, electric field distribution of nanorods dimer, and the corresponding impact on the gold film are studied. It is concluded that the gap size of nanostructures has an exponential correlation with the resonance wavelength. Considering the remarkable influence of the gap size and the surrounding medium environment on the spectral shift of AgNR-film nanostructures, potential applications of the structure as a refractive index sensor and biomolecule measurement are proposed.

In this paper, the finite element method is used to calculate the extinction optical properties and electric field distribution of gold core silver shell nanorod-gold film nanostructures. The volume ratio of silver and the length diameter ratio of core-shell nanorods can be changed by controlling the thickness of silver layer, which can realize the regulation of the longitudinal mode peak position from 600 to 900 nm. The electric field intensity of the core-shell nanorod-gold film structure increases with the increase of the volume ratio of silver. Silver is the plasmon material with the smallest loss in the visible light region, due to its small imaginary part of the dielectric function and lower plasma damping than gold. Therefore, with the increase of the volume ratio of silver, the extinction spectrum intensity of the gold core silver shell nanorod-gold film increases obviously, and the blue shift of the peak position and the narrowing of the full width at half maximum of the wave shape are obvious changes. At the same time, the structure gap confines the light to the deep sub wavelength range, which will produce a hot spot in the gap, which will enhance the electric field.

Based on the coarse-grained UNRES and NARES-2P models of proteins and nucleic acids, respectively, developed in our laboratory, in this work we have developed a coarse-grained model of systems containing proteins and nucleic acids. The UNRES and NARES-2P effective energy functions have been applied to the protein and nucleic-acid components of a system, respectively, while protein-nucleic-acid interactions have been described by the respective coarse-grained potentials developed in our recent work (Yin et al., J. Chem Theory Comput. 2015, 11, 1792). The Debye-Huckel screening has been applied to the electrostatic-interaction energy between the phosphate groups and charged amino-acid side chains. The model has been integrated into the UNRES package for coarse-grained molecular dynamics simulations of proteins and the implementation has been tested for energy conservation in microcanonical molecular dynamics runs and for temperature conservation in canonical molecular dynamics runs. Two case studies were performed: (i) the dynamics of the Ku protein heterodimer bound to DNA, for which it was found that the Ku70/Ku80 protein complex plays an active role in DNA repairing and (ii) conformational changes of the multiple antibiotic resistance (MarA) protein occurring during DNA binding, for which the functionally important motions occurring during this process were identified. © 2018 Wiley Periodicals, Inc.

Grain yields of winter wheat are significantly affected by different straw and nitrogen regimes through regulating root growth and distribution in soil, especially in subsoil layers. In this study, three straw regimes used were S0 (no straw return), S1 (4500 kg ha−1 of maize straw returned), S2 (9000 kg ha−1 of maize straw returned) and four nitrogen regimes used were N0 (no nitrogen applied), N1 (150 kg ha−1 nitrogen applied), N2 (225 kg ha−1 nitrogen applied) and N3 (300 kg ha−1 nitrogen applied). Soil bulk density, soil total porosity, root length density, nitrogen efficiency and grain yield of winter wheat were measured. Straw return could not only decrease the soil bulk density and increase the soil total porosity of farming layer, but also in subsoil layers, especially at plough pan. Straw return treatments enhanced grain yield significantly, which was mainly due to the increase of 1000-kernel weight. S1N2 produced the maximum grain yield, however, S1N1 induced larger subsoil root length density and the proportion of root system in subsoil layers. Compared with S1N2, the grain yield of S1N1 decreased by 4.0%, but nitrogen partial factor productivity (PFPN) and agronomic nitrogen use efficiency (NAE) increased by 44.0% and 32.6%, respectively. Overall, this study demonstrates that suitable amount of straw returned back to field, combining with nitrogen deficiency moderately, maybe a more eco-friendly and sustainable agricultural technique in the field production.

Jimai 22 wheat seeds collected at eight developmental stages [20–38 days after anthesis (daa)] were used to characterize the natural dehydration of wheat seeds. An iTRAQ-based quantitative proteomics technique was applied to identify differentially abundant proteins (DAPs) and evaluate several physiological traits at 20, 26, 32 and 38 daa. Superoxide dismutase and peroxidase activities as well as soluble sugar and soluble protein contents increased during dehydration, while the malondialdehyde content decreased. These results suggested that the antioxidant capacity of seeds increased during the dehydration process. A total of 128 proteins exhibited at least a 1.5-fold difference in abundance relative to the control levels in naturally dehydrating seeds. Forty of the DAPs were related to defense/stress responses. We observed an increase in the abundance of five late embryogenesis abundant proteins, five serpins, a heat shock cognate 70-kDa protein, and the 17.7-kDa Class I heat shock protein, with peak levels detected at 38 daa. Additionally, starch and sucrose metabolic activities were also associated with the natural dehydration of wheat seeds, with trehalose-6-phosphate synthase 7 increasing in abundance during the dehydration process.

Temperature extremes represent an important limiting factor to plant growth and productivity. Low concentration of hydrogen sulfide (H2S) has been proven to function in physiological responses to various stresses. The present study evaluated the effect of foliar application of wheat seedlings with a H2S donor, sodium hydrosulfide (NaHS), on the response to acute heat stress. The results showed that pretreatment with NaHS could promote heat tolerance of wheat seedlings in a dose-dependent manner. Again, it was verified that H2S, rather than other sulfur-containing components or sodion derived from NaHS solution, should contribute to the positive role in promoting wheat seedlings against heat stress. To further study antioxidant mechanisms of NaHS-induced heat tolerance, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities, and H2S, hydrogen peroxide (H2O2), malonaldehyde (MDA), and soluble sugar contents in wheat seedlings were determined. The results showed that, under heat stress, the activities of SOD, CAT, and APX, H2S, H2O2, MDA, and soluble sugar contents in NaHS-pretreated seedlings and its control all increased. Meanwhile, NaHS-pretreated seedlings showed higher antioxidant enzymes activities and gene expression levels as well as the H2S and soluble sugar levels, and lower H2O2, MDA contents induced by heat stress. While little effect was detected in antioxidant enzymes activities and soluble substances contents in pretreated wheat seedlings compared with its control under normal culture conditions (data not shown). All of our results suggested that exogenous NaHS could alleviate oxidative damage and improve heat tolerance by regulating the antioxidant system in wheat seedlings under heat stress.

Small cell lung cancer (SCLC) is a highly lethal malignancy with the 5-year survival rate of less than 7%. Chemotherapy-resistance is a major challenge for SCLC treatment in clinic. In the study, we developed a high-throughput drug screen strategy to identify new drugs that can enhance the sensitivity of chemo-drug cisplatin in SCLC. This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as a sensitizer of cisplatin. Further study validated that auranofin synergistically enhanced the anti-tumor activity of cisplatin in chemo-resistant SCLC cells, which was accompanied by the enhanced induction of cell cycle arrest and apoptosis. The synergistic action of auranofin and cisplatin was through ROS overproduction, thereby leading to mitochondrial dysfunction and DNA damage. Furthermore, in vivo study demonstrated that the combination treatment of auranofin and cisplatin dramatically inhibited tumor growth in SCLC. Therefore, our study provides a rational basis for further clinical study to test whether auranofin could enhance the sensitivity of cisplatin-based therapy in SCLC patients.

ObjectivesTo develop a model of in-hospital mortality using medical record front page (MRFP) data and assess its validity in case-mix standardisation by comparison with a model developed using the complete medical record data.DesignA nationally representative retrospective study.SettingRepresentative hospitals in China, covering 161 hospitals in modelling cohort and 156 hospitals in validation cohort.ParticipantsRepresentative patients admitted for acute myocardial infarction. 8370 patients in modelling cohort and 9704 patients in validation cohort.Primary outcome measuresIn-hospital mortality, which was defined explicitly as death that occurred during hospitalisation, and the hospital-level risk standardised mortality rate (RSMR).ResultsA total of 14 variables were included in the model predicting in-hospital mortality based on MRFP data, with the area under receiver operating characteristic curve of 0.78 among modelling cohort and 0.79 among validation cohort. The median of absolute difference between the hospital RSMR predicted by hierarchical generalised linear models established based on MRFP data and complete medical record data, which was built as ‘reference model’, was 0.08% (10th and 90th percentiles: −1.8% and 1.6%). In the regression model comparing the RSMR between two models, the slope and intercept of the regression equation is 0.90 and 0.007 in modelling cohort, while 0.85 and 0.010 in validation cohort, which indicated that the evaluation capability from two models were very similar.ConclusionsThe models based on MRFP data showed good discrimination and calibration capability, as well as similar risk prediction effect in comparison with the model based on complete medical record data, which proved that MRFP data could be suitable for risk adjustment in hospital performance measurement.

Summary: Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed 3-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template-based modeling methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes. Availability and Implementation: Freely available on the web at http://www.unres.pl/ . Contact: has5@cornell.edu

It is well established that the RpoN-RpoS sigma factor (σ 54 -σ S ) cascade plays an essential role in differential gene expression during the enzootic cycle of Borrelia burgdorferi , the causative agent of Lyme disease. The RpoN-RpoS pathway is activated by the response regulator/σ 54 -dependent activator (also called bacterial enhancer-binding protein [bEBP]) Rrp2. One unique feature of Rrp2 is that this activator is essential for cell replication, whereas RpoN-RpoS is dispensable for bacterial growth. How Rrp2 controls cell replication, a function that is independent of RpoN-RpoS, remains to be elucidated. In this study, by generating a series of conditional rrp2 mutant strains, we demonstrated that the N-terminal receiver domain of Rrp2 is required for spirochetal growth. Furthermore, a D52A point mutation at the phosphorylation site within the N terminus of Rrp2 abolished cell replication. Mutation of the ATPase motif within the central domain of Rrp2 did not affect spirochetal replication, indicating that phosphorylation-dependent ATPase activity of Rrp2 for σ 54 activation is not required for cell growth. However, deletion of the C-terminal domain or a 16-amino-acid truncation of the helix-turn-helix (HTH) DNA-binding motif within the C-terminal domain of Rrp2 abolished spirochetal replication. It was shown that constitutive expression of rpoS is deleterious to borrelial growth. We showed that the essential nature of Rrp2 is not due to an effect on rpoS . These data suggest that phosphorylation-dependent oligomerization and DNA binding of Rrp2 likely function as a repressor, independently of the activation of σ 54 , controlling an essential step of cell replication in B. burgdorferi . IMPORTANCE Bacterial enhancer-binding proteins (bEBPs) are a unique group of transcriptional activators specifically required for σ 54 -dependent gene transcription. This work demonstrates that the B. burgdorferi bEBP, Rrp2, has an additional function that is independent of σ 54 , that of its essentiality for spirochetal growth, and such a function is dependent on its N-terminal signal domain and C-terminal DNA-binding domain. These findings expand our knowledge on bEBP and provide a foundation to further study the underlying mechanism of this new function of bEBP.

Effects of exogenous abscisic acid (ABA) on antioxidant enzymes activities and endosperm cell division of two wheat (Triticum aestivum L.) cultivars were investigated. Results showed that the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity in flag leaves of both cultivars is elevated by application of ABA and it is correlated with less membrane damage: lower malondialdehyde (MDA). Exogenous ABA significantly increased endosperm cell number and endosperm division rate, finally increased grain weight. Although ABA treatment decreased endogenous zeatin riboside (ZR) content in flag leaves from 7 to 28 days after anthesis (DAA), indole acetic acid (IAA) levels were significantly increased by spraying with ABA. Correlation analysis showed that endogenous contents of ZR, ABA, and IAA in grains were positively and significantly correlated with grain-filling rate. IAA content in leaves was positively and significantly correlated with grain-filling rate. The results suggested that increased grain weight of ABA-treated plants was due to higher antioxidant abilities of flag leaf resulting in longer maintenance of photosynthetic capacity and higher grain-filling rate.

Based on the coarse-grained UNRES and NARES-2P models of proteins and nucleic acids, respectively, developed in our laboratory, in this work we have developed a coarse-grained model of systems containing proteins and nucleic acids. The UNRES and NARES-2P effective energy functions have been applied to the protein and nucleic-acid components of a system, respectively, while protein-nucleic-acid interactions have been described by the respective coarse-grained potentials developed in our recent work (Yin et al., J. Chem Theory Comput. 2015, 11, 1792). The Debye-Hückel screening has been applied to the electrostatic-interaction energy between the phosphate groups and charged amino-acid side chains. The model has been integrated into the UNRES package for coarse-grained molecular dynamics simulations of proteins and the implementation has been tested for energy conservation in microcanonical molecular dynamics runs and for temperature conservation in canonical molecular dynamics runs. Two case studies were performed: (i) the dynamics of the Ku protein heterodimer bound to DNA, for which it was found that the Ku70/Ku80 protein complex plays an active role in DNA repairing and (ii) conformational changes of the multiple antibiotic resistance (MarA) protein occurring during DNA binding, for which the functionally important motions occurring during this process were identified. © 2018 Wiley Periodicals, Inc.

The relationship between protein sequence and structure arises entirely from amino acid physical properties. An alternative method is therefore proposed to identify homologs in which residue equivalence is based exclusively on the pairwise physical property similarities of sequences. This approach, the property factor method (PFM), is entirely different from those in current use. A comparison is made between our method and PSI BLAST. We demonstrate that traditionally defined sequence similarity can be very low for pairs of sequences (which therefore cannot be identified using PSI BLAST), but similarity of physical property distributions results in almost identical 3D structures. The performance of PFM is shown to be better than that of PSI BLAST when sequence matching is comparable, based on a comparison using targets from CASP10 (89 targets) and CASP11 (51 targets). It is also shown that PFM outperforms PSI BLAST in informatically challenging targets.

Severe water deficit (SD) severely limited the photo-assimilate supply during the grain-filling stages. Although the ethylene and polyamines (PAs) have been identified as important signaling molecules involved in stress tolerance, it is yet unclear how 1-Aminocylopropane-1-carboxylic acid (ACC) and PA biosynthesis involving wheat abdominal phloem characters mitigate SD-induced filling inhibition. The results obtained indicated that the SD down-regulated the TaSUT1 expression and decreased the activities of sucrose synthase (SuSase, EC2.4.1.13), ADP glucose pyrophosphorylase (AGPase, EC2.7.7.27), soluble starch synthase (SSSase, EC2.4.1.21), then substantially limited grain filling. As a result, increased ACC and putrescine (Put) concentrations and their biosynthesis-related gene expression reduced spermidine (Spd) biosynthesis under SD condition. And, the ACC and PA biosynthesis in inferior grains was more sensitive to SD than that in superior grains. Intermediary cells (ICs) of caryopsis emerged prematurely under SD to compensate for the weakened photo-assimilate transport functions of sieve elements (SEs). Finally, plasmolysis and nuclear chromatin condensation of phloem parenchyma cells (PPC) and membrane degradation of SEs, as well as the decreased ATPase activity on plasma membranes of ICs and PPC at the later filling stage under SD were responsible for the considerably decreased weight of inferior grains.

We recently introduced a physically based approach to sequence comparison, the property factor method (PFM). In the present work, we apply the PFM approach to the study of a challenging set of sequences-the bacterial chemotaxis protein CheY, the N-terminal receiver domain of the nitrogen regulation protein NT-NtrC, and the sporulation response regulator Spo0F. These are all response regulators involved in signal transduction. Despite functional similarity and structural homology, they exhibit low sequence identity. PFM sequence comparison demonstrates a statistically significant qualitative difference between the sequence of CheY and those of the other two proteins that is not found using conventional alignment methods. This difference is shown to be consonant with structural characteristics, using distance matrix comparisons. We also demonstrate that residues participating strongly in native contacts during unfolding are distributed differently in CheY than in the other two proteins. The PFM result is also in accord with dynamic simulation results of several types. Molecular dynamics simulations of all three proteins were carried out at several temperatures, and it is shown that the dynamics of CheY are predicted to differ from those of NT-NtrC and Spo0F. The predicted dynamic properties of the three proteins are in good agreement with experimentally determined B factors and with fluctuations predicted by the Gaussian network model. We pinpoint the differences between the PFM and traditional sequence comparisons and discuss the informatic basis for the ability of the PFM approach to detect physical differences between these sequences that are not apparent from traditional alignment-based comparison.

Two wheat cultivars (Triticum aestivum L.) were used to evaluate the effects of post-anthesis severe water deficit (SD) on starch content and granule size distribution and their relations with ethylene and spermidine (Spd). Comparison to the well-watered (WW) treatment, SD led to lower Spd and higher 1-aminocylopropane-1-carboxylic acid (ACC) concentrations and ethylene evolution rate (EER) in grains at the critical stage of forming starch granules. Application of Spd or aminoethoxyvinylglycine (AVG) significantly reduced ACC concentration and EER and increased Spd concentration, while ethephon or methylglyoxal-bis (MGBG) had an opposite impact. The volume and surface area distribution of starch granules showed a bimodal curve, while the number distribution exhibited a unimodal curve. SD caused a marked drop in grain weight, grain number and starch content, also led to a significant reduction in the proportion (both by volume and by surface area) of B-type starch granules (10 μm). Application of Spd or AVG increased the proportion (both by volume and by surface area) of B-type starch granules under SD. Correlation analysis suggested that ethylene and Spd showed an antagonism relation in the formation of B-type granules. These results suggested that it would be good for the formation of B-type starch granules to have the physiological traits of higher Spd and lower ACC concentrations and ethylene emission under SD.

Lodging is an important constraint limiting wheat yields and quality by bending or breaking stems on wheat (Triticum aestivum L.) production worldwide. This study was conducted to determine whether lignin accumulation and lodging resistance of winter wheat could be affected by application of paclobutrazol (PP333) or gibberellin acid (GA3) at stem elongation stage (DC 3.0). Two winter wheat cultivars, Jimai22 (lodging-tolerant cultivar; JM22) and Shannong16 (lodging-susceptible cultivar; SN16), were used to investigate the effect of exogenous hormones on wheat lodging behavior, lignin accumulation in culms and its relation to lodging resistance. Results showed that application of PP333 significantly reduced plant height and the length of basal second internode, while GA3 had the opposite effect. Lignin accumulation was positively and significantly correlated with the breaking strength of internode and culm lodging resistance index (CLRI). PP333 significantly increased lignin accumulation and its related enzymes activities in the basal second internode, and also increased the diameter, wall thickness, filling degree of the internode, thus the breaking strength and lodging resistance of wheat increased. Whereas, GA3 caused opposite effect on those characteristics. These results suggested that the exogenous PP333 or GA3 application significantly changed the risk of lodging occurred not only by altering the plant height but also by altering the physical strength of the basal part of the culm internode, especially, altering the lignin accumulation and its related enzymes activities in basal internode.

The compositions of tillers, which are directly related to the grain yield, markedly affect the structure and quality of a wheat population. Constructing a rational population structure based on the superior tillers also enhances the yield potential of wheat. However, information on the non-uniformity in the productive capacity of tillers in wheat and the lack of effective measures to regulate the population structure is insufficient. In the present study, two winter wheat cultivars, Shannong15 (SN15) and Shannong8355 (SN8355), were used to (1) evaluate the differences in productive capacity among tillers in wheat, (2) analyze the underlying mechanisms between endogenous hormone and wheat tiller production by exogenous hormones, and (3) investigate the effects of exogenous hormones on the grain yield of wheat. Results showed that low-position tillers (early initiated tillers) exhibited a higher yield per spike than high-position tillers (late-initiated tillers) in both cultivars. Cluster analysis showed that tillers of SN15 were classified into two groups: the superior tiller group, which included the main stem (0), the first primary tiller (I), and the second primary tiller (II); and the inferior tiller group, which included the third primary tiller (III) and the first secondary tiller (I-p). The tillers of SN8355 were classified into three groups: superior tiller group (0 and I), intermediate tiller group (II and III) and inferior tiller group (I-p). Indole-3-acetic acid (IAA) and zeatin (ZT) exhibited important functions in regulating the growth of tillers. Exogenous hormones regulated the growth of wheat tiller buds by affecting the contents of IAA and ZT as well as the ratios of IAA to ZT and abscisic acid (ABA) to ZT in the tiller nodes, thereby regulating the growth of tillers. Furthermore, the external application of IAA, gibberellic acid (GA3), and ABA evidently reduced the number of stems per square meter at wintering and jointing stages, which could promote the accumulation of single-stem biological yield. The wheat plants in the IAA, GA3, and ABA groups yielded more grains and higher grain weight than those in the control group; as a result, grain yield increased. These results suggested that the tiller composition of a wheat population could be restructured (based on the superior tiller) by inhibiting the growth of inferior tillers induced by external IAA, GA, and ABA treatments to enhance the yield potential of wheat.

This study was to test the hypothesis that polyamines (PAs) and ethylene may be involved in mediating the effect of water deficit on grain filling. Two wheat cultivars, drought-tolerant Shannong16 (SN16) and drought-sensitive Jimai22 (JM22), were used and subjected to well-watered and severe water deficit (SD) during grain filling. SD reduced the weight of superior and inferior grains, by 7.38 and 23.54 % in JM22, 13.8 and 2.2 % in SN16, respectively. Higher free-spermidine (Spd) and free-spermine (Spm) concentration and lower free-putrescine (Put) concentration, ethylene evolution rate (EER) and 1-aminocylopropane-1-carboxylic acid (ACC) concentration were found in superior grains than those in inferior ones. Opposite to the variations of Spd and Spm concentration, ACC, Put concentration and EER were significantly increased under SD. The percentage variation of PAs and ACC differed with cultivars and grain types. ACC concentration of superior and inferior grains under SD increased significantly at 21 days post-anthesis, by 90 and 164 % in JM22, 65 and 13.2 % in SN16, respectively. The equivalent value of Put concentration was 1.04 and 7.9 % in JM22, 34.4 and 10.3 % in SN16. Spd concentration of superior grains showed a higher decrease than that of inferior ones in both cultivars, while Spm exhibited an opposite trend between both grain types. These percentage variations were highly consistent with the differed responses of weight of both grain types to SD in JM22 and SN16. Grain filling rate was negatively correlated with EER and ACC concentration, while positively correlated with Spd and Spm concentration as well as the ratio of Spd or Spm to ACC. Exogenous Spd or aminoethoxyvinylglycine (an inhibitor of ethylene synthesis by inhibiting ACC synthesis) obviously reduced ACC concentration and EER and increased Spd and Spm concentration, while exogenous ethephon (an ethylene-releasing agent) or methylglyoxal-bis (an inhibitor of Spd and Spm synthesis) showed the opposite effects. The results suggested that it would be good for wheat to have the physiological traits of higher Spd and Spm, as well as a higher Spd/ACC or Spm/ACC, under SD.

Granule size distribution of wheat starch is an important characteristic that can affect its chemical composition and the functionality of wheat products. Two high-yield winter wheat cultivars were used to evaluate the effects of the application of exogenous ABA or GA during the reproductive phase of the initial grain filling on starch granule size distribution and starch components in grains at maturity. The results indicated that a bimodal curve was found in the volume and surface area distribution of grain starch granules, and a unimodal curve was observed for the number distribution under all treatments. The exogenous ABA resulted in a significant increase in the proportions (both by volume and by surface area) of B-type (9.9 μm) starch granules, while, the exogenous GA3 led to converse effects on size distribution of those starch granules. The exogenous ABA also increased starch, amylose and amylopectin contents at maturity but significantly reduced the ratio of amylose to amylopectin. Application of GA3 significantly reduced starch content, amylopectin content but increased the ratio of amylose to amylopectin. The ratio of amylose to amylopectin showed a significant and negative relationship with the volume proportion of granules

In a field experiment, two winter wheat (Triticum aestivum L.) cultivars, Tainong 18 (a large-spike cultivar) and Jinan 17 (a multiple-spike cultivar), were treated with 78% (S1), 50% (S2), and 10% (S3) of full sunshine (S0, control) from anthesis to maturity to determine the responses of photosynthetic characteristics and antioxidative enzyme activities in a flag leaf. Compared with S0 treatment, the chlorophyll (Chl) content and maximal efficiency of photosystem II (PSII) photochemistry (Fv/Fm) of flag leaves were enhanced in treatments S1 and S2. From 0 to 7 d post flowering, the Chl content and Fv/Fm in S3 were also higher than those in S0, but significantly lower than those in controls, respectively. With the increase of shading intensity, the effective quantum yield of PSII (ΦPSII) was promoted; whereas, the ratio of Chl a/b declined. Compared with S0, treatments S2 and S3 significantly suppressed the activities of superoxide dismutase (SOD) and peroxidase (POD), net photosynthetic rate (P N), and contents of total soluble sugar, nevertheless, S1 treatment showed positive effects on the above parameters. Under the same shading condition, Jinan 17 had larger Chl content and higher activities of PSII and antioxidative enzymes, but lower malondialdehyde (MDA) content than Tainong 18. The results indicated that multiple-spike cultivar was more advantageous for the Huang-Huai-Hai Plain, where shading problem occurs later during the growth period, than the large-spike cultivar, because of the lesser damage in a flag leaf and better photosynthetic function of the former one. Wheat plants under S1 shading condition had relatively high activities of antioxidative enzymes and a low degree of membrane lipid peroxidation, which was in favor of stress resistance, maintaining high P N duration, and accumulation of photosynthates in wheat plants.

Nitric oxide (NO) is a key signaling molecule in different physiological processes of plants, including programmed cell death (PCD). PCD of tillers plays an important role in surviving which are major components of grain yield. PCD was triggered in wheat leaves of main stem and tillers by NO content under different nitrogen treatments. In wheat, NO could be synthesized endogenously by nitrate reductase (NR). As an inducible enzyme, NR activity was closely related to substrate concentration. Therefore, different nitrogen levels would change NR activity and NO production. The objective of this study was to determine the effects of NR activity, NO production, and the correlation between them on different tillers growth, development, senescence, and kernel protein content under different nitrogen levels. Field-experiments were conducted in 2009–2011 growing seasons, using two wheat cultivars with different spike-types. Results showed that for main stem and primary tillers, NR activity and NO content reached high level at heading stage, while for secondary tiller, the level of NR activity was low, but NO content was high in the present research. The NO synthesis depending on NR activity in wheat leaves was significant in the early growing stage, but the NO synthesis weakened with the progress of growing period. NO was related to the senescence of wheat leaves, but PCD was more sensitive to marked changes of NO content than NO content itself. N application had marked influence on the aging process of primary tiller, while had little influence on that of main stem and secondary tiller. Moreover, N fertilizer application could increase spike rate and protein content of primary tiller by N fertilizer application.

Increasing plant density is one of the most efficient ways of increasing wheat (Triticum aestivum L.) grain production. However, overly dense plant populations have an increased risk of lodging. We examined lignin deposition during wheat stem development and the regulatory effects of plant density using the wheat cultivars shannong23 and weimai8. Plants were cultivated at densities of 75, 225 and 375 plants per m2 during two growing seasons. Our results showed that decreasing plant density enhanced culm quality, as revealed by increased culm diameter, wall thickness and dry weight per unit length, and improved the structure of sclerenchyma and vascular bundles by increasing lignification. In addition, more lignins were deposited in the secondary cell walls, resulting in strong lodging resistance. The guaiacyl unit was the major component of lignin and there was a higher content of the syringyl unit than that of the hydroxybenzyl unit. Furthermore, we hypothesised that the syringyl unit may correlate with stem stiffness. We describe here, to the best of our knowledge, the systematic study of the mechanism involved in the regulation of stem breaking strength by plant density, particularly the effect of plant density on lignin biosynthesis and its relationship with lodging resistance in wheat.

Stay-green, a key trait of wheat, can not only increase the yield of wheat but also its resistance to heat stress during active photosynthesis. Cytokinins are the most potent general coordinator between the stay-green trait and senescence. The objectives of the present study were to identify and assess the effects of cytokinins on the photosynthetic organ and heat resistance in wheat. Two winter wheat cultivars, Wennong 6 (a stay-green cultivar) and Jimai 20 (a control cultivar), were subjected to heat stress treatment from 1 to 5 days after anthesis (DAA). The two cultivars were sprayed daily with 10 mg L-1 of 6-benzylaminopurine (6-BA) between 1 and 3 DAA under ambient and elevated temperature conditions. We found that the heat stress significantly decreased the number of kernels per spike and the grain yield (P < 0.05). Heat stress also decreased the zeatin riboside (ZR) content, but increased the gibberellin (GA3), indole-3-acetic acid (IAA), and abscisic acid (ABA) contents at 3 to 15 DAA. Application of 6-BA significantly (P < 0.05) increased the grain-filling rate, endosperm cell division rate, endosperm cell number, and 1,000-grain weight under heated condition. 6-BA application increased ZR and IAA contents at 3 to 28 DAA, but decreased GA3 and ABA contents. The contents of ZR, ABA, and IAA in kernels were positively and significantly correlated with the grain-filling rate (P < 0.05), whereas GA3 was counter-productive at 3 to 15 DAA. These results suggest that the decrease in grain yield under heat stress was due to a lower ZR content and a higher GA3 content compared to that at elevated temperature during the early development of the kernels, which resulted in less kernel number and lower grain-filling rate. The results also provide essential information for further utilization of the cytokinin substances in the cultivation of heat-resistant wheat.

Ba 1.0 Sr 1.0 FeO 4+ δ (BSFO) with A 2 BO 4 structure as a cathode material for intermediate temperature solid oxide fuel cells (IT-SOFCs) is synthesized through an ethylene diamine tetraacetic acid (EDTA)–citrate process, and characterized by X-ray diffraction. Field emission scanning electron microscopy shows that BSFO cathode is well attached to the La 0.9 Sr 0.1 Ga 0.8 Mg 0.2 O 3− δ (LSGM) electrolyte. The electrical conductivity measured by DC four-probe method increases as the temperature increases. A linear relationship between ln( σT ) and 1000/ T indicates that the conducting behavior obeys the small polaron conductivity mechanism. Electrochemical performance of BSFO cathode on LSGM electrolyte is investigated in the temperature range from 500 °C to 800 °C. The results indicate that oxygen adsorption/dissociation process dominates cathodic reaction. Furthermore, the polarization resistance of BSFO cathode decreases with increasing temperature, and declines to 1.42 Ω cm 2 at 800 °C. These results show that BSFO can be a promising cathode material used on LSGM electrolyte for IT-SOFCs.

The objective of this study was to understand the morphological, physiological, and molecular responses of wheat roots to nitrate supply at seedling stage. Two wheat genotypes, Jimai 22 and Shannong 15, were grown in Hoagland's nutrient solution with different nitrate levels at seedling stage. Results indicated that the plant dry weight and N accumulation increased with the increase of nitrate supply. The number of axial root, total uptake area (TUA), and active uptake area (AUA) increased with more nitrate supply. Correlation analysis indicated that significant positive correlations existed between N accumulation and dry weight, N accumulation and AUA, and N accumulation and AUA/TUA. Although, the expressions of NRT2.1, NRT2.2, and NRT2.3 decreased with nitrate supply increased, the expressions of NRT1, NRT2.1, and NRT2.3 could maintain high level at N3 treatment. The free amino acid and NO3− content in shoot also increased with the increased nitrate application, but no significant difference was found in root among the treatments. These results implied that the increase of N uptake by nitrate supply was due to the morphological and physiological responses of wheat roots and the high expression level of TaNRT genes. Similarly, the contribution of morphological, physiological, and molecular parameters was different between two genotypes of wheat.