A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing.

FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.

Co-fermentation of the main sugar types from a beechwood organosolv hydrolysate by several strains of Bacillus coagulans results in effective lactic acid production.

Bacillus coagulans is an interesting facultative anaerobic microorganism for biotechnological production of lactic acid that arouses interest. To determine the efficiency of biotechnological production of lactic acid from lignocellulosic feedstock hydrolysates, five Bacillus coagulans strains were grown in lignocellulose organosolv hydrolysate from ethanol/water-pulped beechwood. Parameter estimation based on a Monod-type model was used to derive the basic key parameters for a performance evaluation of the batch process. Three of the Bacillus coagulans strains, including DSM No. 2314, were able to produce lactate, primarily via uptake of glucose and xylose. Two other strains were identified as having the ability of utilizing cellobiose to a high degree, but they also had a lower affinity to xylose. The lactate yield concentration varied from 79.4 ±â€¯2.1 g/L to 93.7 ±â€¯1.4 g/L (85.4 ±â€¯4.7 % of consumed carbohydrates) from the diluted organosolv hydrolysate.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box.

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

Cytonuclear Epistasis Controls the Density of Symbiont Wolbachia pipientis in Nongonadal Tissues of Mosquito Culex quinquefasciatus.

Wolbachia pipientis, a bacterial symbiont infecting arthropods and nematodes, is vertically transmitted through the female germline and manipulates its host's reproduction to favor infected females. Wolbachia also infects somatic tissues where it can cause nonreproductive phenotypes in its host, including resistance to viral pathogens. Wolbachia-mediated phenotypes are strongly associated with the density of Wolbachia in host tissues. Little is known, however, about how Wolbachia density is regulated in native or heterologous hosts. Here, we measure the broad-sense heritability of Wolbachia density among families in field populations of the mosquito Culex pipiens, and show that densities in ovary and nongonadal tissues of females in the same family are not correlated, suggesting that Wolbachia density is determined by distinct mechanisms in the two tissues. Using introgression analysis between two different strains of the closely related species C. quinquefasciatus, we show that Wolbachia densities in ovary tissues are determined primarily by cytoplasmic genotype, while densities in nongonadal tissues are determined by both cytoplasmic and nuclear genotypes and their epistatic interactions. Quantitative-trait-locus mapping identified two major-effect quantitative-trait loci in the C. quinquefasciatus genome explaining a combined 23% of variance in Wolbachia density, specifically in nongonadal tissues. A better understanding of how Wolbachia density is regulated will provide insights into how Wolbachia density can vary spatiotemporally in insect populations, leading to changes in Wolbachia-mediated phenotypes such as viral pathogen resistance.

A brief dataset on the model-based evaluation of the growth performance of Bacillus coagulans and l-lactic acid production in a lignin-supplemented medium.

The data presented in this article are related to the research article entitled "Model-based characterization of growth performance and l-lactic acid production with high optical purity by thermophilic Bacillus coagulans in a lignin-supplemented mixed substrate medium (R. Glaser and J. Venus, 2016) [1]". This data survey provides the information on characterization of three Bacillus coagulans strains. Information on cofermentation of lignocellulose-related sugars in lignin-containing media is given. Basic characterization data are supported by optical-density high-throughput screening and parameter adjustment to logistic growth models. Lab scale fermentation procedures are examined by model adjustment of a Monod kinetics-based growth model. Lignin consumption is analyzed using the data on decolorization of a lignin-supplemented minimal medium.

Model-based characterisation of growth performance and l-lactic acid production with high optical purity by thermophilic Bacillus coagulans in a lignin-supplemented mixed substrate medium.

Three Bacillus coagulans strains were characterised in terms of their ability to grow in lignin-containing fermentation media and to consume the lignocellulose-related sugars glucose, xylose, and arabinose. An optical-density high-throughput screening was used for precharacterisation by means of different mathematical models for comparison (Logistic, Gompertz, Baranyi, Richards & Stannard, and Schnute). The growth response was characterised by the maximum growth rate and lag time. For a comparison of the screening and fermentation results, an unstructured mathematical model was proposed to characterise the lactate production, bacterial growth and substrate consumption. The growth model was then applied to fermentation procedures using wheat straw hydrolysates. The results indicated that the unstructured growth model can be used to evaluate lactate producing fermentation. Under the experimental fermentation conditions, one strain showed the ability to tolerate a high lignin concentration (2.5g/L) but lacked the capacity for sufficient pentose uptake. The lactate yield of the strains that were able to consume all sugar fractions of glucose, xylose and arabinose was ∼83.4%. A photometric measurement at 280nm revealed a dynamic change in alkali-lignin concentrations during lactate producing fermentation. A test of decolourisation of vanillin, ferulic acid, and alkali-lignin samples also showed the decolourisation performance of the B. coagulans strains under study.

Unraveling the Structure-Affinity Relationship between Cucurbit[n]urils (n = 7, 8) and Cationic Diamondoids.

We report the measurement of the binding constants (Ka) for cucurbit[n]uril (n = 7, 8) toward four series of guests based on 2,6-disubstituted adamantanes, 4,9-disubstituted diamantanes, 1,6-disubstituted diamantanes, and 1-substituted adamantane ammonium ions by direct and competitive 1H NMR spectroscopy. Compared to the affinity of CB[7]·Diam(NMe3)2, the adamantane diammonium ion complexes (e.g., CB[7]·2,6-Ad(NH3)2 and CB[7]·2,6-Ad(NMe3)2) are less effective at realizing the potential 1000-fold enhancement in affinity due to ion-dipole interactions at the second ureidyl C═O portal. Comparative crystallographic investigation of CB[7]·Diam(NMe3)2, CB[7]·DiamNMe3, and CB[7]·1-AdNMe3 revealed that the preferred geometry positions the +NMe3 groups ≈0.32 Å above the C═O portal; the observed 0.80 Å spacing observed for CB[7]·Diam(NMe3)2 reflects the simultaneous geometrical constraints of CH2···O═C close contacts at both portals. Remarkably, the CB[8]·IsoDiam(NHMe2)2 complex displays femtomolar binding affinity, placing it firmly alongside the CB[7]·Diam(NMe3)2 complex. Primary or quaternary ammonium ion looping strategies lead to larger increases in binding affinity for CB[8] than for CB[7], which we attribute to the larger size of the carbonyl portals of CB[8]; this suggests routes to develop CB[8] as the tightest binding host in the CB[n] family. We report that alkyl group fluorination (e.g., CB[7]·1-AdNH2Et versus CB[7]·1-AdNH2CH2CF3) does not result in the expected increase in Ka value. Finally, we discuss the role of solvation in nonempirical quantum mechanical computational methodology, which is used to estimate the relative changes in Gibbs binding free energies.

A Nexus between Theory and Experiment: Non-Empirical Quantum Mechanical Computational Methodology Applied to Cucurbit[n]uril⋠Guest Binding Interactions.

A training set of eleven X-ray structures determined for biomimetic complexes between cucurbit[n]uril (CB[7 or 8]) hosts and adamantane-/diamantane ammonium/aminium guests were studied with DFT-D3 quantum mechanical computational methods to afford ΔGcalcd binding energies. A novel feature of this work is that the fidelity of the BLYP-D3/def2-TZVPP choice of DFT functional was proven by comparison with more accurate methods. For the first time, the CB[n]⋅guest complex binding energy subcomponents [for example, ΔEdispersion , ΔEelectrostatic , ΔGsolvation , binding entropy (-TΔS), and induced fit Edeformation(host) , Edeformation(guest) ] were calculated. Only a few weeks of computation time per complex were required by using this protocol. The deformation (stiffness) and solvation properties (with emphasis on cavity desolvation) of cucurbit[n]uril (n=5, 6, 7, 8) isolated host molecules were also explored by means of the DFT-D3 method. A high ρ2 =0.84 correlation coefficient between ΔGexptl and ΔGcalcd was achieved without any scaling of the calculated terms (at 298 K). This linear dependence was utilized for ΔGcalcd predictions of new complexes. The nature of binding, including the role of high energy water molecules, was also studied. The utility of introduction of tethered [-(CH2 )n NH3 ]+ amino loops attached to N,N-dimethyl-adamantane-1-amine and N,N,N',N'-tetramethyl diamantane-4,9-diamine skeletons (both from an experimental and a theoretical perspective) is presented here as a promising tool for the achievement of new ultra-high binding guests to CB[7] hosts. Predictions of not yet measured equilibrium constants are presented herein.

Enzyme-based lignocellulose hydrolyzation--Sauter mean diameter of raw materials as a basis for cellulase performance characterization and yield prediction.

The characterization of cellulase performance for industrial-scale processes holds special challenges. A huge gap exists between the enzyme characterization in a laboratory and large-scale process performances. Common cellulase mixtures from Novozymes and enzymes provided by Moscow State University were used to hydrolyze wheat straw, grass, pine wood, and aspen wood. Glucose yields from the enzymatic hydrolysis of the raw materials were investigated as a function of cellulase enzyme loading and of particle sizes with different solid loading. The particle size had a significant effect on glucose yield, while the used enzyme concentration had a much smaller effect. Hydrolyses of sets of wheat straw particles were used to introduce a substrate-specific kinetic enzyme unit. The data were also used to generate an empirical model in order to predict the glucose yield on the basis of the Sauter mean diameter of the feedstocks.

Influence of hydrophobic residues on the binding of CB[7] toward diammonium ions of common ammonium···ammonium distance.

We report the binding constants of CB[7] toward a series of naphthalene diammonium and 4,4'-dipiperidinium derivatives and compare the results with those obtained previously for CB[7]·3b by (1)H NMR and X-ray crystallography. The nature of binding in the host·guest complexes was investigated using quantum mechanical tools.

Enzyme-based lignocellulose hydrolyzation - Brief data survey for cellulase performance characterization on behalf of the Sauter mean diameter of raw material particles.

The data presented here supports the informational background of enzyme-based lignocellulose hydrolyzation, cellulase characterization, and sugar yield prediction for the work "Enzyme-based lignocellulose hydrolyzation - Sauter mean diameter of raw materials as a basis for cellulase performance characterization and yield prediction" by Glaser [1]. Glucose yields from the enzymatic hydrolysis of the raw materials were shown as a function of cellulase enzyme loading as well as of particle size with different solid loading. The data for the proposed methods of the determination of enzyme activity in inhomogeneous samples of lignocellulosic raw materials are presented. The data of the empirical model that was developed for the prediction of hydrolysis yields for different enzyme concentrations, substrate specific particle size, and solid loadings, are given. Data are also given in relation of terms of scale-up opportunities.

Somatic Wolbachia (Rickettsiales: Rickettsiaceae) levels in Culex quinquefasciatus and Culex pipiens (Diptera: Culicidae) and resistance to West Nile virus infection.

The endosymbiotic bacteria Wolbachia pipientis Hertig infects a wide variety of insect species and can increase viral resistance in its host. Wolbachia naturally infects Culex quinquefasciatus Say and Culex pipiens L. mosquitoes, both vectors of West Nile virus (WNV). We recently demonstrated that Wolbachia infection of Cx. quinquefasciatus laboratory strain Ben95 increases host resistance to WNV infection, reducing vector competence. This observation raised the possibility that Wolbachia could impact vector competence in other populations of Cx. quinquefasciatus or Cx. pipiens. To investigate this possibility, Wolbachia densities were measured in Ben95 Cx. quinquefasciatus and compared with densities in a newly established colony of Cx. quinquefasciatus, and in field-collected and colonized Cx. pipiens. Wolbachia densities in somatic tissues of Ben95 Cx. quinquefasciatus were significantly higher than densities in the other mosquito populations tested. There was also no significant spatiotemporal variation in Wolbachia density in the field-collected Cx. pipiens, although significant familial differences were observed. Correlating Wolbachia densities and vector competence in individual colonized Cx. pipiens indicated that the densities of somatic Wolbachia observed in the mosquitoes other than Ben95 Cx. quinquefasciatus were too low to inhibit WNV infection and reduce vector competence. These results suggest that the high Wolbachia densities capable of inducing resistance to WNV in Ben95 Cx. quinquefasciatus are not a general characteristic of Cx. quinquefasciatus or Cx. pipiens mosquitoes and that the impact of Wolbachia on vector competence in field populations of Cx. quinquefasciatus and Cx. pipiens, if any, is likely to be limited to specific populations.

Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation.

Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes.

Cucurbit[7]uril⋠guest pair with an attomolar dissociation constant.

Host⋅guest complexes between cucurbit[7] (CB[7]) or CB[8] and diamantane diammonium ion guests 3 or 6 were studied by (1) H NMR spectroscopy and X-ray crystallography. (1) H NMR competition experiments revealed that CB[7]⋅6 is among the tightest monovalent non-covalent complexes ever reported in water with Ka =7.2×10(17) M(-1) in pure D2 O and 1.9×10(15) M(-1) in D2 O buffered with NaO2 CCD3 (50 mM). The crystal structure of CB[7]⋅6 allowed us to identify some of the structural features responsible for the ultratight binding, including the distance between the NMe3 (+) groups of 6 (7.78 Å), which allows it to establish 14 optimal ion-dipole interactions with CB[7], the complementarity of the convex van der Waals surface contours of 6 with the corresponding concave surfaces of CB[7], desolvation of the CO portals within the CB[7]⋅6 complex, and the co-linearity of the C7  axis of CB[7] with the N(+) ⋅⋅⋅N(+) line in 6. This work further blurs the lines of distinction between natural and synthetic receptors.

Screening of Bacillus coagulans strains in lignin supplemented minimal medium with high throughput turbidity measurements.

The aim of this study was to extend the options for screening and characterization of microorganism through kinetic growth parameters. In order to obtain data, automated turbidimetric measurements were accomplished to observe the response of strains of Bacillus coagulans. For the characterization, it was decided to examine the influence of varying concentrations of lignin with respect to bacterial growth. Different mathematical models are used for comparison: logistic, Gompertz, Baranyi and Richards and Stannard. The growth response was characterized by parameters like maximum growth rate, maximum population, and the lag time. In this short analysis we present a mathematical approach towards a comparison of different microorganisms. Furthermore, it can be demonstrated that lignin in low concentrations can have a positive influence on the growth of B. coagulans.

Solution- and solid-state conformations of C(α)-alkyl analogues of methylphenidate (Ritalin) salts: avoidance of gauche(+)gauche(-) interactions.

Alkyl analogues of methylphenidate (Ritalin) salts are slow onset, long duration dopamine reuptake inhibitors with a potential use as a cocaine abuse pharmacotherapy. X-ray crystallographic studies and nuclear magnetic resonance (NMR) investigations strongly suggest that avoidance of sterically unfavorable gauche(-)gauche(+) orientations effectively influences both the C(α)-alkyl side chain conformation and the formation of a predominant rotamer about the CH-CH bond ligating piperidine and C(Ar)R moieties. The favored CH-CH rotamer in D(2)O and in CD(2)Cl(2) of the pharmacologically interesting i-Bu and CH(2)-cyc-Pnt (RS,RS)-salts has the same antiperiplanar arrangement that was found in the crystal structures, although there clearly is a fast equilibrium involving smaller amounts of synclinal partners. While the rotamer in the (RS,SR)-i-Bu HCl crystal structure exhibits a synclinal orientation for the vicinal pair of adjacent methine protons, the weighted time-averaged arrangement for these protons becomes almost completely antiperiplanar when the crystals are dissolved in D(2)O. Increased steric congestion around the CH-CH bond in the analogous N-methyl tertiary ammonium salts seems to augment the quantity of the preferred rotamer within the mixture. The stereochemistry of the species observed via NMR seems to arise from specific combinations of N-methyl orientation and avoidance of sterically unfavorable gauche(-)gauche(+) arrangements.

The native Wolbachia endosymbionts of Drosophila melanogaster and Culex quinquefasciatus increase host resistance to West Nile virus infection.

BACKGROUND: The bacterial endosymbiont Wolbachia pipientis has been shown to increase host resistance to viral infection in native Drosophila hosts and in the normally Wolbachia-free heterologous host Aedes aegypti when infected by Wolbachia from Drosophila melanogaster or Aedes albopictus. Wolbachia infection has not yet been demonstrated to increase viral resistance in a native Wolbachia-mosquito host system. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated Wolbachia-induced resistance to West Nile virus (WNV; Flaviviridae) by measuring infection susceptibility in Wolbachia-infected and Wolbachia-free D. melanogaster and Culex quinquefasciatus, a natural mosquito vector of WNV. Wolbachia infection of D. melanogaster induces strong resistance to WNV infection. Wolbachia-infected flies had a 500-fold higher ID50 for WNV and produced 100,000-fold lower virus titers compared to flies lacking Wolbachia. The resistance phenotype was transmitted as a maternal, cytoplasmic factor and was fully reverted in flies cured of Wolbachia. Wolbachia infection had much less effect on the susceptibility of D. melanogaster to Chikungunya (Togaviridae) and La Crosse (Bunyaviridae) viruses. Wolbachia also induces resistance to WNV infection in Cx. quinquefasciatus. While Wolbachia had no effect on the overall rate of peroral infection by WNV, Wolbachia-infected mosquitoes produced lower virus titers and had 2 to 3-fold lower rates of virus transmission compared to mosquitoes lacking Wolbachia. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that Wolbachia can increase resistance to arbovirus infection resulting in decreased virus transmission in a native Wolbachia-mosquito system. The results suggest that Wolbachia reduces vector competence in Cx. quinquefasciatus, and potentially in other Wolbachia-infected mosquito vectors.

Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D (1)H and (13)C NMR spectroscopy. This fraction with a molar mass of 2.39x10(5)g mol(-1) comprised D- and L-Gal, D-Glc, D-Xyl, D-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1-->2)- or (1-->4)-linked d-xylopyranosyl, (1-->3)-linked L-galactopyranosyl, (1-->3)-linked D-glucopyranosyl, and (1-->3)-linked D-glucopyranosyluronic acid and comprises a possible acidic building unit: [(2 or 4)-beta-D-Xylp-(L-->3)]m-alpha-D-Glcp-(1-->3)-alpha-D-GLCPA-(1-->3)-L-Galp(l-->. Attached to the backbone are sulfate groups and nonreducing terminal D-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.

The Drosophila protein palmitoylome: characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases.

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization,RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627 and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2)and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.

To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of approximately 25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response.