Low Inhibitor Concentrations Research Articles

A Cardiolipin-Deficient Mutant of Rhodobacter Sphaeroides has an Altered Cell Shape and is Impaired in Biofilm Formation

Cell shape has been suggested to play an important role in regulating bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase deletion mutant of the rod-shaped bacterium Rhodobacter sphaeroides—in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%—produces ellipsoidal-shaped cells that are impaired in forming biofilms. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroidescells with a low concentration (10 μg/mL) of the small molecule MreB inhibitor, S-(3,4-dichlorobenzyl) isothiourea (A22), produced ellipsoid-shaped cells that had no obvious growth defect, yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of this bacterium to adapt to its environment.

Neurite outgrowth is driven by actin polymerization even in the presence of actin polymerization inhibitors.

Actin polymerization is a universal mechanism to drive plasma membrane protrusion in motile cells. One apparent exception to this rule is continuing, or even accelerated outgrowth of neuronal processes in the presence of actin polymerization inhibitors. This fact together with a key role of microtubule dynamics in neurite outgrowth led to the concept that microtubules directly drive plasma membrane protrusion, either in the course of polymerization or motor-driven sliding. Surprisingly, a possibility that unextinguished actin polymerization drives neurite outgrowth in the presence of actin drugs was not explored. We show that cultured hippocampal neurons treated with cytochalasin D or latrunculin B contained dense accumulations of branched actin filaments at ∼50% of neurite tips at all tested drug concentrations (1-10 μM). Actin polymerization was required for neurite outgrowth, because only low concentrations of either inhibitor increased the length and/or a number of neurites, whereas high concentrations inhibited neurite outgrowth. Importantly, neurites undergoing active elongation invariably contained a bright F-actin patch at the tip, whereas actin-depleted neurites never elongated, even though they still contained dynamic microtubules. Stabilization of microtubules by taxol treatment did not stop elongation of cytochalasin d-treated neurites. We conclude that actin polymerization is indispensable for neurite elongation.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

Improvement of sugar yields from corn stover using sequential hot water pretreatment and disk milling

Efficient pretreatment is essential for economic conversion of lignocellulosic feedstocks into monosaccharides for biofuel production. To realize high sugar yields with low inhibitor concentrations, hot water or dilute acid pretreatment followed by disk milling is proposed. Corn stover at 20% solids was pretreated with hot water at 160–200°C for 4–8min with and without subsequent milling. Hot water pretreatment and disk milling acted synergistically to improve glucose and xylose yields by 89% and 134%, respectively, compared to hot water pretreatment alone. Hot water pretreated (180°C for 4min) and milled samples had the highest glucose and xylose yields among all hot water pretreated and milled samples, which were comparable to samples pretreated with 0.55% dilute acid at 160°C for 4min. However, samples pretreated with 1% dilute acid at 150°C for 4min and disk milled had the highest observed glucose (87.3%) and xylose yields (83.4%).

Fluorescence properties and sequestration of peripheral anionic site specific ligands in bile acid hosts: Effect on acetylcholinesterase inhibition activity

The increase in fluorescence intensity of model acetyl cholinesterase (AChE) inhibitors like propidium iodide (PI) and ethidium bromide (EB) is due to sequestration of the probes in primary micellar aggregates of bile acid (BA) host medium with moderate binding affinity of ca. 102–103M−1. Multiple regression analysis of solvent dependent fluorescence behavior of PI indicates the decrease in total nonradiative decay rate due to partial shielding of the probe from hydrogen bond donation ability of the aqueous medium in bile acid bound fraction. Both PI and EB affects AChE activity through mixed inhibition and consistent with one site binding model; however, PI (IC50=20±1μM) shows greater inhibition in comparison with EB (IC50=40±3μM) possibly due to stronger interaction with enzyme active site. The potency of AChE inhibition for both the compounds is drastically reduced in the presence of bile acid due to the formation of BA-inhibitor complex and subsequent reduction of active inhibitor fraction in the medium. Although the inhibition mechanism still remains the same, the course of catalytic reaction critically depends on equilibrium binding among several species present in the solution; particularly at low inhibitor concentration. All the kinetic parameters for enzyme inhibition reaction are nicely correlated with the association constant for BA-inhibitor complex formation.

Mechanism of inhibition and decoupling of oxygen evolution from electron transfer in photosystem II by fluoride, ammonia and acetate

Ca2+ extraction from oxygen-evolving complex (OEC) of photosystem II (PSII) is accompanied by decoupling of oxygen evolution/electron transfer processes [Semin et al. Photosynth. Res. 98 (2008) 235] and appearance of a broad EPR signal at g=2 (split “S3” signal) what can imply the relationship between these effects. Split signal have been observed not only in Ca-depleted PSII but also in PSII membranes treated by fluoride anions, sodium acetate, and NH4Cl. Here we investigated the question: can such compounds induce the decoupling effect during treatment of PSII like Ca2+ extraction does? We found that F−, sodium acetate, and NH4Cl inhibit O2 evolution in PSII membranes more effectively than the reduction of artificial electron acceptor 2,6-dichlorophenolindophenol, i.e. the action of these compounds is accompanied by decoupling of these processes in OEC. Similarity of effects observed after Ca2+ extraction and F−, CH3COO− or NH4Cl treatments suggests that these compounds can inactivate function of Ca2+. Such inactivation could originate from disturbance of the network of functionally active hydrogen bonds around OEC formed with participation of Ca2+. This inhibition effect is observed in the region of low concentration of inhibitors. Increasing of inhibitor concentration is accompanied by appearance of other sites of inhibition.

Revisiting direct electron transfer in nanostructured carbon laccase oxygen cathodes

The biocatalytic electroreduction of oxygen has been studied on large surface area graphite and Vulcan® carbon electrodes with adsorbed Trametes trogii laccase. The electrokinetics of the O2 reduction reaction (ORR) was studied at different electrode potentials, O2 partial pressures and concentrations of hydrogen peroxide.Even though the overpotential at 0.25mA·cm−2 for the ORR at T1Cu of the adsorbed laccase on carbon is 0.8V lower than for Pt of similar geometric area, the rate of the reaction and thus the operative current density is limited by the enzyme reaction rate at the T2/T3 cluster site for the adsorbed enzyme. The transition potential for the rate determining step from the direct electron transfer (DET) to the enzyme reaction shifts to higher potentials at higher oxygen partial pressure.Hydrogen peroxide produced by the ORR on bare carbon support participates in an inhibition mechanism, with uncompetitive predominance at high H2O2 concentration, non-competitive contribution can be detected at low inhibitor concentration.

Techno-economic analysis of the deacetylation and disk refining process: characterizing the effect of refining energy and enzyme usage on minimum sugar selling price and minimum ethanol selling price.

BackgroundA novel, highly efficient deacetylation and disk refining (DDR) process to liberate fermentable sugars from biomass was recently developed at the National Renewable Energy Laboratory (NREL). The DDR process consists of a mild, dilute alkaline deacetylation step followed by low-energy-consumption disk refining. The DDR corn stover substrates achieved high process sugar conversion yields, at low to modest enzyme loadings, and also produced high sugar concentration syrups at high initial insoluble solid loadings. The sugar syrups derived from corn stover are highly fermentable due to low concentrations of fermentation inhibitors. The objective of this work is to evaluate the economic feasibility of the DDR process through a techno-economic analysis (TEA).ResultsA large array of experiments designed using a response surface methodology was carried out to investigate the two major cost-driven operational parameters of the novel DDR process: refining energy and enzyme loadings. The boundary conditions for refining energy (128–468 kWh/ODMT), cellulase (Novozyme’s CTec3) loading (11.6–28.4 mg total protein/g of cellulose), and hemicellulase (Novozyme’s HTec3) loading (0–5 mg total protein/g of cellulose) were chosen to cover the most commercially practical operating conditions. The sugar and ethanol yields were modeled with good adequacy, showing a positive linear correlation between those yields and refining energy and enzyme loadings. The ethanol yields ranged from 77 to 89 gallons/ODMT of corn stover. The minimum sugar selling price (MSSP) ranged from $0.191 to $0.212 per lb of 50 % concentrated monomeric sugars, while the minimum ethanol selling price (MESP) ranged from $2.24 to $2.54 per gallon of ethanol.ConclusionsThe DDR process concept is evaluated for economic feasibility through TEA. The MSSP and MESP of the DDR process falls within a range similar to that found with the deacetylation/dilute acid pretreatment process modeled in NREL’s 2011 design report. The DDR process is a much simpler process that requires less capital and maintenance costs when compared to conventional chemical pretreatments with pressure vessels. As a result, we feel the DDR process should be considered as an option for future biorefineries with great potential to be more cost-effective.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0358-0) contains supplementary material, which is available to authorized users.

Compensatory role of the Nrf2–ARE pathway against paraquat toxicity: Relevance of 26S proteasome activity

Oxidative stress and the ubiquitin–proteasome system play a key role in the pathogenesis of Parkinson disease. Although the herbicide paraquat is an environmental factor that is involved in the etiology of Parkinson disease, the role of 26S proteasome in paraquat toxicity remains to be determined. Using PC12 cells overexpressing a fluorescent protein fused to the proteasome degradation signal, we report here that paraquat yielded an inhibitory effect on 26S proteasome activity without an obvious decline in 20S proteasome activity. Relative low concentrations of proteasome inhibitors caused the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is targeted to the ubiquitin–proteasome system, and activated the antioxidant response element (ARE)-dependent transcription. Paraquat also upregulated the protein level of Nrf2 without increased expression of Nrf2 mRNA, and activated the Nrf2–ARE pathway. Consequently, paraquat induced expression of Nrf2-dependent ARE-driven genes, such as γ-glutamylcysteine synthetase, catalase, and hemeoxygenase-1. Knockdown of Nrf2 or inhibition of γ-glutamylcysteine synthetase and catalase exacerbated paraquat-induced toxicity, whereas suppression of hemeoxygenase-1 did not. These data indicate that the compensatory activation of the Nrf2–ARE pathway via inhibition of 26S proteasome serves as part of a cellular defense mechanism to protect against paraquat toxicity.

Hemicellulose-derived sugars solubilisation of rape straw. Cofermentation of pentoses and hexoses by Escherichia coli

Bioconversion of hemicellulose sugars is essential for increasing fuel ethanol yields from lignocellulosic biomass. We report for the first time with rape straw, bioethanol production from hemicellulose sugars. Rape straw was pretreated at mild conditions with sulfuric acid to solubilize the hemicellulose fraction. This pretreatment allows obtaining a prehydrolysate, consisting basically in a solution of monomeric hemicellulosic sugars, with low inhibitor concentrations. The remaining water insoluble solid constitutes a cellulose-enriched, free of extractives material. The influence of temperature (120ºC and 130ºC), acid concentration (2-4% w/v) and pretreatment time (30-180 min) on hemicellulose-derived sugars solubilisation was evaluated. The highest hemicellulosic sugars recovery, 72.3%, was achieved at 130ºC with 2% sulfuric acid and 60 min. At these conditions, a concentrated sugars solution, 52.4 g/L, was obtained after three acid consecutive contacts, with 67% xylose and acetic acid concentration above 4.5 g/L. After a detoxification step by activated charcoal or ion-exchange resin, prehydrolysate was fermented by ethanologenic Escherichia coli. An alcoholic solution of 25 g/L and 86% of theoretical ethanol yield was attained after 144 h when the prehydrolysate was detoxified by ion-exchange resin. The results obtained in the present work show sulfuric acid pretreatment under mild conditions and E. coli as an interesting process to exploit hemicellulosic sugars in rape straw.

A Cardiolipin-Deficient Mutant of Rhodobacter sphaeroides Has an Altered Cell Shape and Is Impaired in Biofilm Formation

Cell shape has been suggested to play an important role in the regulation of bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase (Δcls) mutant of the rod-shaped bacterium Rhodobacter sphaeroides--in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%--produces ellipsoid-shaped cells that are impaired in biofilm formation. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 μg/ml) of the small-molecule MreB inhibitor S-(3,4-dichlorobenzyl)isothiourea produced ellipsoid-shaped cells that had no obvious growth defect yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of the bacterium to adapt to its environment. Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in the regulation of R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.

English

The abundance of second generation feedstock reinforces the consideration of biofuel over fossil fuel, as bioethanol can be produced from lignocellulosic materials. However, the pretreatment required for oxidation of lignocellulose into hexose often results in the production of inhibitors likely to impede the activity of Saccharomyces cerevisiae during bioethanol production. This study aimed to investigate the comparative inhibitory effects of acetic acid and vanillin on the viability of S. cerevisiae and the production yield of bioethanol. Different concentrations of inhibitors were spiked in the fermentation broth then the production of bioethanol monitored overtime and correlated with cell viability. The results showed that the inhibition of S. cerevisiae by vanillin is more potent compared to acetic acid; however the reduction of bioethanol yield after 12 h was more pronounced with acetic acid (42.8% reduction) than with vanillin (33.3% reduction) which was ascribed to the simultaneous production of weak acids during the fermentation process. The viability test has shown that in the presence of lower concentrations of inhibitors, S. cerevisiae can adapt for the first 12 h of fermentation and then may improve ethanol production yield overtime. At lower concentrations (2 g/l vanillin and 4 g/l acetic acid) the effect of inhibitors on the viability of S. cerevisiae and ethanol productivity does not last and can be overcome by the adaptation of the yeast. However, the presence of higher concentrations (4 g/l vanillin and 6 g/l acetic acid) results to nearly total inhibition of bioethanol production and the remediation of such effect may therefore require a detoxification process. Key words: Bioethanol production, Saccharomyces cerevisiae, inhibition, acetic acid, vanillin, cell viability.

Current progress in high cell density yeast bioprocesses for bioethanol production.

High capital costs and low reaction rates are major challenges for establishment of fermentation-based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.

Conversion of Sweet Sorghum Bagasse Residue to Glucose by Dilute Acid Hydrolysis

In the present work, hydrolysis of sweet sorghum bagasse residue using dilute acid was investigated. The sweet sorghum bagasse residue was from pretreatment. Process parameters investigated include reactor temperature, reaction time, and acid concentration in order to find suitable conditions for hydrolysis of the cellulose fraction. The effects of dilute acid hydrolysis were assessed by glucose yield and decomposition products concentration. The highest yield of glucose, 70.10%, was found at the temperature of 170°C, acid concentration of 1.0% (w/w), and a reaction time of 10 min. Meanwhile, 0.99 g/L of acetic acid, 0.22 g/L of furfural, 0.73 g/L of hydro-xymethyl furfural, and 1.60 g/L of levulinic acid were obtained under these conditions, which concluded that the decomposition of glucose was slight and confirmed the selectivity of this dilute acid hydrolysis of the sweet sorghum bagasse residue. The present work aims to take full advantage of the sweet sorghum bagasse residue and obtain glucose solutions with a low concentration of growth inhibitors.

Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation

Human megakaryocytes release glutamate and express glutamate-gated Ca2+-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca2+ responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca2+ flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca2+ entry into Meg-01 cells, amplified Ca2+ responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5–100μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100μM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20μM and 36μM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation.In conclusion, NMDARs provide a novel pathway for Ca2+ entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.

Optimization and kinetic analysis on the sulfuric acid – Catalyzed depolymerization of wheat straw

The objectives of this work were to optimize the experimental condition and to study the kinetic behavior of wheat straw depolymerization with sulfuric acid (2wt%, 3wt%, and 4wt%) at different temperatures (120°C, 130°C, and 140°C). The two-fraction kinetic model was obtained for the prediction of the generations of product and by-product during depolymerization. The kinetic parameters of the two-fraction model were analyzed using an Arrhenius-type equation. Applying the kinetic two-fraction model, the optimum condition for wheat straw depolymerization was 3wt% H2SO4 at 130°C for 75min, which yielded a high concentration of fermentable sugars (xylose 8.934g/L, glucose 1.363g/L, and arabinose 1.203g/L) and low concentrations of microbial inhibitors (furfural 0.526g/L and acetic acid 1.192g/L). These results suggest that the model obtained in this study can satisfactorily describe the formation of degradation products and the depolymerization mechanism of wheat straw.

The Role of Butyrylcholinesterase in Beta‐Amyloid Formation in Neuroblastoma Cells

The progression of Alzheimer's disease (AD) correlates with changes in the level of cholinesterase activity, i.e., butyrylcholinesterase (BuChE) increases while acetylcholinesterase (AChE) decreases. The consequence is a decrease in the level of acetylcholine and loss of cognitive function. In addition, both enzymes are associated with neurotoxic β‐amyloid (Aβ) plaques. As such, butyrylcholinestersase inhibitors are a potential therapeutic to relieve the symptoms of AD. The BuChE specific irreversible inhibitor di‐n‐butyl 2‐chlorophenyl phosphate (DBPP) was tested for its effect on the formation of β‐amyloid and the amyloid precursor protein (APP) by ELISA and Western Blot analysis, respectively. Results of the ELISA assay showed an increase in Aβ‐40 and 42 concentration in neuroblastoma cells treated with 10‐5 M DBPP. In contrast lower. concentrations of 10‐6 M and 10‐8 M DBPP induced a lower concentration of Aβ‐40 and 42 when compared to the control.. Western Blot analysis of the cell lysate supernatant showed a decrease APP formation with 10‐5 DBPP. but little or no difference at the lower concentrations of inhibitor. Supported in part by CSUPERB and the CSULB Mini Grant Program.

ABT‐737 Inhibits Full Length And Cleaved Pro‐Apoptotic Bcl‐xL, Resulting in Differential Effects on Death And Survival

ABT-737 is a pharmacological inhibitor of the anti-apoptotic Bcl-2 family protein, Bcl-xL. ABT-737 occupies the BH3-binding domain, supporting apoptosis of cancer cells. We have shown that ABT-737 lowers neuronal metabolic efficiency by inhibiting ATP synthase activity. However, we unexpectedly found that ABT-737 protects brains from ischemic injury in vivo. In our recent study, ABT-737 inhibited formation and function of the cleavage product of full length Bcl-xL, pro-apoptotic ΔN-Bcl-xL. In the current study, we find that high concentration ABT-737 blocks the effects of full length Bcl-xL but low concentration ABT-737 inhibits ΔN-Bcl-xL; this suggests that cleavage of full length Bcl-xL during cell death stimuli exposes the ABT-737 binding site. We tested whether ABT-737 exerts neuroprotective properties by altering mitochondrial membrane permeability. We found that pre-treatment of neurons with 1μM ABT-737 aggravated death induced by glutamate. We observed a loss of mitochondrial potential and a decline in ATP production in ABT-737 and glutamate treated cells. In contrast, pre-treatment with 10nM ABT-737, a hundred fold lower concentration, rescued neurons from death and augmented mitochondrial potential and ATP production during glutamate challenge. Incubation of recombinant ΔN-Bcl-xL protein with isolated mitochondria significantly reduced mitochondrial potential, rescued by CsA and low dose ABT-737, suggesting involvement of mitochondrial permeability transition pore. Our findings suggest that low concentrations of Bcl-xL inhibitors may promote survival by preferentially binding to the pro-apoptotic form of Bcl-xL even as high concentrations potentiate cell death in malignant cells.

Blocking integrin inactivation as an anti-angiogenic therapy.

During angiogenesis, endothelial cell migration is coordinated by integrin‐mediated contact with the extra‐cellular matrix (ECM), coupled with receptor tyrosine kinase signalling to regulate dynamic cytoskeletal and plasma membrane reorganization. A recent paper by Vitorino et al (2015) defined a new MAP4K4–moesin–talin–β1‐integrin pathway that could be therapeutically exploited to suppress pathologic angiogenesis.

Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.