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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.

Straw return is an important management tool for tackling and promoting soil nutrient conservation and improving crop yield in Huang-Huai-Hai Plain, China. Although the incorporation of maize straw with deep plowing and rotary tillage practices are widespread in the region, only few studies have focused on rotation tillage. To determine the effects of maize straw return on the nitrogen (N) efficiency and grain yield of winter wheat (Triticum aestivum L.), we conducted experiments in this region for 3 years. Five treatments were tested: (i) rotary tillage without straw return (RT); (ii) deep plowing tillage without straw return (DT); (iii) rotary tillage with total straw return (RS); (iv) deep plowing tillage with total straw return (DS); (v) rotary tillage of 2 years and deep plowing tillage in the 3rd year with total straw return (TS). Treatments with straw return increased kernels no. ear−1, thousand-kernel weight (TKW), grain yields, ratio of dry matter accumulation post-anthesis, and nitrogen (N) efficiency whereas reduced the ears no. ha−1 in the 2011–2012 and 2012–2013 growing seasons. Compared with the rotary tillage, deep plowing tillage significantly increased the grain yield, yield components, total dry matter accumulation, and N efficiency in 2013–2014. RS had significantly higher straw N distribution, soil inorganic nitrogen content, and soil enzymes activities in the 0–10 cm soil layer compared with the DS and TS. However, significantly lower values were observed in the 10–20 and 20–30 cm soil layers. TS obtained approximately equal grain yield as DS, and it also reduced the resource costs. Therefore, we conclude that TS is the most economical method for increasing grain yield and N efficiency of winter wheat in Huang-Huai-Hai Plain.

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.

1,25(OH)(2)D(3) has already been reported to function in some diseases. However, its role in hyperlipidemia (HLP) remains unknown. This study aims to investigate the effect of 1,25(OH)(2)D(3) on HLP rats. Rat models were established by high-fat diet feeding, perfusion of different doses of 1,25-(OH)(2)D(3) and injection of TGF-β1 siRNA. Whole blood viscosity, plasma viscosity, hematocrit, and erythrocyte aggregation index were detected, together with levels of biochemical indexes, 6-keto-PGF1α, and TXB2 in serum. Levels of oxidative stress indexes and inflammatory factors in serum and liver tissues were determined. TGF-β1 and Smad3 expression in serum, liver tissues, and aorta was detected. 1,25(OH)(2)D(3) lowered HLP-induced rise of whole blood viscosity, red blood cell aggregation index, plasma viscosity, and hematocrit, TC, TG, LDL-C, apoB, ALT, AST, TXB2, MDA, IL-1β, TNF-α, and increased HLP-induced decrease of HDL-C, apoAI, 6-keto-PGF1α, SOD, GSH-Px, CAT, and T-AOC. TGF-β1 and Smad3 expression in serum, liver tissue, and aorta of 1,25(OH)(2)D(3)-treated rats reduced. High 1,25(OH)(2)D(3) dose and inhibited TGF-β/Smad signaling pathway alleviated lipid metabolism, liver function, and atherosclerotic injury in HLP rats. Our study found that 1,25(OH)(2)D(3) improves blood lipid metabolism, liver function, and atherosclerosis injury by constraining the TGF-β/Smad signaling pathway in rats with HLP.

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.

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.