PARTICIPATION OF EXTRACELLULAR NUCLEOTIDES IN THE WOUND RESPONSE OF DASYCLADUS VERMICULARIS AND ACETABULARIA ACETABULUM (DASYCLADALES,CHLOROPHYTA)1

As assayed by fluorescent reporter dyes, nitric oxide (NO) and H₂O₂, two downstream signaling agents induced by wounding in the alga Dasycladus vermicularis (Scop.) Krasser, can also be induced in unwounded Dasycladus cells by μM Adenosine 5′[γ‐thio]triphosphate (ATPγS) and Adenosine 5′‐[β‐thio]diphosphate (ADPβS), but not by Adenosine 5′‐O‐thiomonophosphate (AMPS). These nucleotide‐induced responses are blocked by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an antagonist of animal purinoceptors, and by adenosine, a feed‐back inhibitor of extracellular nucleotide responses in animals. Similar nucleotide‐ and nucleotide‐antagonist responses were observed in Acetabularia acetabulum (L.) P. C. Silva. Significant levels of ATP released from Dasycladus cells were measured at wound sites by a sensitive luciferin‐luciferase assay. Additionally, the normal wound‐induced production of NO and H₂O₂ in Dasycladus can be blocked by pretreating the cells with PPADS. Our results indicate that nucleotides released from wounds can serve as a signal to trigger wound responses in algae, and that coordinated signaling between extracellular nucleotides and the NO pathway may have been established early during the evolution of plants.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Hypoxia stimulates vesicular ATP release from rat osteoblasts

Many neuronal and non‐neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y₁ receptor, stimulate osteoclast formation and activity, whilst P2Y₂ receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation‐dependent manner, with mature, bone‐forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N‐ethylmaleimide (1–1,000 µM) inhibited basal ATP release by up to 99%. The presence of granular ATP‐filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O₂) for up to 3 min increased ATP release from osteoblasts up to 2.5‐fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 µM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N‐ethylmaleimide (100 µM) attenuated the hypoxia‐induced ATP release by up to 80%. Depletion of quinacrine‐stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O₂ could influence local purinergic signaling and affect the balance between bone formation and bone resorption. J. Cell. Physiol. 220: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Potential application of the nisin Z preparation of Lactococcus lactis W8 in preservation of milk

Aims: The aim of the study is to evaluate the effectiveness of the preparation of nisin Z from Lactococcus lactis W8‐fermented milk in controlling the growth of spoilage bacteria in pasteurized milk. Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and 15°C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria were found to have the ability to survive pasteurization temperature. Except Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim milk or fat milk inoculated with each of the spoilage bacteria reduced the initial counts (about 5 log CFU ml^?1) to an undetectable level within 8–20 h. The nisin Z preparation extended the shelf life of milk to 2 months under refrigeration. Conclusions: The nisin Z preparation from L. lactis W8‐fermented milk was found to be effective as a backup preservative to counteract postpasteurization contamination in milk. Significance and Impact of the Study: A rapid inhibition of spoilage bacteria in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is more significant in comparison with the commercially available nisin (nisin A). The nisin Z preparation can be used instead of commercial nisin, which is not effective in fat milk.     

Synergistic antibacterial activity of the essential oil of Cuminum cyminum L. seed and nisin in a food model

Aims: To investigate effects of various concentrations of the essential oil of Cuminum cyminum L. seed alone and in combination with nisin on survival of vegetative forms of Bacillus cereus and Bacillus subtilis in a food model (commercial barley soup) and their ultrastructure. Methods and Results: Gas chromatography–mass spectrometry analysis indicated that cumin aldehyde (29·02%) and α‐terpinen‐7‐al (20·70%) constituted the highest amount of the essential oil. The lowest concentration of the essential oil significantly affected the growth of the bacteria at 8°C but not at 25°C. Synergistic effect of the essential oil in combination with the lowest concentration of nisin was observed on the bacteria at 8°C. Evaluation of the sensory properties showed that concentration of 0·15 μl ml^?1 of the essential oil was the most acceptable. Conclusions: The essential oil of C. cyminum L. seed showed the most bactericidal effects on B. cereus at 8°C. Ultrastructural studies of vegetative cells confirmed the synergistic destructive effects of the essential oil and nisin on membrane and cell wall of the bacteria. Significance and Impact of the Study: Utilization of essential oil of C. cyminum L. seed in combination with nisin can inhibit growth of food‐borne pathogens in food.     

Oxygen deficiency and its effect on the adenylate system in Acetobacter in the submerse acetic fermentation

Summary It is well known that Acetobacter is extremely sensitive in high total concentrations (GK)¹ of ethanol and acetic acid. In the acetator, at a total concentration (GK) of 13%, ATP pool and growth show reverse behaviour. During the stationary, acidifying phase, the extracellular adenylate concentration amounts to 70% of the total edenylate pool (AN=ATP+ADP+AMP). In this range, the average value of the intracellular energy charge [EC=(ATP+1/2ADP)/(ATP+ADP+AMP)] is 0.82.After 45 s of interruption of aeration, the EC of the total culture dropped to a value of 0.58. After several weeks of storage, the EC of the inoculum amounted to 0.50.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Isolation of sensitive nisin-sensing GFP<Subscript>uv</Subscript> bioassay <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains using FACS

Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP_uv nisin bioassay. It harbors the GFP_uv encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP_uv fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml⁻¹) for nisin than the parent strain (10 pg ml⁻¹). This showed that mutants of LAC275 could successfully be isolated using FACS.     

Cardiomyocyte death induced by ischaemic/hypoxic stress is differentially affected by distinct purinergic P2 receptors

Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown. In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischaemic/hypoxic stress. Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N₂ for 16 hrs in a glucose‐free medium. In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone‐associated‐DNA‐fragments, a marker of apoptosis. Addition of either apyrase, which degrades extracellular ATP, or various inhibitors of ATP release via connexin hemichannels fully abolished ischaemic/hypoxic stress‐associated apoptosis. To dissect the role of specific P2 receptor subtypes, we used a combined approach: (i) non‐selective and, when available, subtype‐selective P2 antagonists, were added to cardiomyocytes before ischaemic/hypoxic stress; (ii) selected P2 receptors genes were silenced via specific small interfering RNAs. Both approaches indicated that the P2Y₂ and P2χ₇ receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y₄ receptor has a protective effect. Overall, these findings indicate a role for ATP and its receptors in modulating cardiomyocyte damage during ischaemic/hypoxic stress.     

Attachment of lactic acid bacteria to beef-muscle surfaces

The effect of immersion time and cell concentration in the attachment of several lactic acid bacteria with antibacterial activity to beef-muscle surface was studied. The number of firmly attached bacteria increased with immersion time in the case ofPediococcus acidilacti, Lactobacillus sake, Lactococcus cremoris (two strains) andPediococcus acidilacti. Pediococcus pentosaceus, Lactococcus lactis andLactobacillus curvatus reached maximum adhesion after 15–30 min. The highest strength of attachment (Sr values) were observed after 15–30 min of contact, time except forP. pentosaceus. For all strains, the number of bacteria adhering to meat increased with increasing cell concentration in the adhesion medium. The highest strength of attachment was observed at a cell concentration of 10⁵/mL mainly forL. sake, L. lactis andL. cremoris. Due to their attachment characteristics,L. sake, L. lactis andL. cremoris are proposed as potential biocontrol agents because they could grow on meat surface and limit the potential attachment of pathogenic microorganisms.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against influenza virus infection

Aims: To evaluate the protective effects of oral administration of milk fermented with a Lactococcus strain against influenza virus (IFV) infection in a mouse model. Methods and Results: Milk fermented with exopolysaccharide‐producing Lactococcus lactis subsp. cremoris (L. cremoris) FC was orally administered to BALB/c mice for 12 days. Mice were intranasally infected with IFV A/New Caledonia/20/99 (H1N1) on day 8, and survival was determined for 14 days after IFV infection. Survival rate and body weight loss after IFV infection in the L. cremoris FC fermented milk‐administered group were significantly improved compared with those in the control group. In the unfermented milk‐administered group, survival rate was not improved, whereas body weight loss was slightly improved compared with that in the control group. The mean virus titre in the lung of the L. cremoris FC fermented milk‐administered group 3 days after infection was significantly decreased compared with that in the control group. Conclusions: These results suggest that oral administration of milk fermented with L. cremoris FC protects mice against IFV infection. Significance and Impact of the Study: These results demonstrate that oral administration of milk fermented with exopolysaccharide‐producing Lactococcus strains might protect host animals against IFV infection.     

Solvation structure of sodium chloride (Na+–Cl-) ion pair in acetonitrile (AN)–dimethyl formamide (DMF) mixtures

Solvation structures of Na⁺–Cl^? ion pair are investigated in acetonitrile (AN)–dimethylformamide (DMF) isodielectric mixtures. The potentials of mean force of Na⁺–Cl^? in the five compositions of mixtures show minima corresponding to a contact ion pair (CIP) and a solvent-shared ion pair (SShIP). The solvent-separated ion pair minima are present in lower mole fractions of AN (x_AN ≤ 0.50). CIPs are found to be more stable than the SShIPs. From a thermodynamic decomposition of the potentials of mean force, we find that the formation of the ion pair is entropically driven in these compositions. The most stable CIP is in pure AN. The local solvation structures around the ion pair are analysed through the running coordination numbers, excess coordination numbers, solvent orientational distributions and density profiles. We find that both Na⁺ and Cl^? are preferentially solvated by DMF.     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

Immunomodulatory efficacy of nisin—a bacterial lantibiotic peptide

Nisin is a peptide bacteriocin, grouped under the category of lantibiotics. It is naturally produced by Lactococcus lactis to eliminate other competing gram‐positive bacteria from its vicinity. Moreover under certain conditions it is reported to be effective against a broad range of gram‐negative bacteria as well. Thus, it has been widely used as a safe food preservative especially in the dairy industry. Because of its wide‐scale consumption, its effect on eukaryotic cells should be of great concern. Here we examine the immunomodulatory efficacy of nisin in vitro. MTT‐based cytotoxicity assay demonstrated nisin's cytotoxicity on human T‐cell lymphoma Jurkat cells, Molt‐4 cells and freshly cultured human lymphocytes at over 200 µM concentration (IC₅₀225 µM ). The cell death mechanism induced by nisin in all these lymphocyte types was independent of oligonucleosomal DNA fragmentation, as analyzed by agarose gel electrophoresis and comet assay. Additionally, scanning electron microscope and fluorescence microscopy demonstrated the ability of nisin to activate human PMNs in vitro. Nisin‐activated neutrophils extruded intact nuclear chromatin to form NETs, well known for neutralization of virulence factors and extermination of bacterial pathogens. Nisin's presence also elevated neutrophil intracellular superoxide levels, normally produced by activated NADPH oxidase and prerequisite to NET formation. These nisin‐induced responses in cellular representatives of two separate branches of human immune system—adaptive and innate—although leading to cell death, did not include DNA fragmentation. From these findings, we propose that nisin might trigger similar AICD mechanisms in lymphocytes and neutrophils, different from conventional apoptosis which involves DNA fragmentation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Tissue layer specific regulation of leaf length and width in Arabidopsis as revealed by the cell autonomous action of ANGUSTIFOLIA

In vascular plants the shoot apical meristem consists of three tissue layers, L1, L2 and the L3, that are kept separate during organ formation and give rise to the epidermis (L1) and the subepidermal tissues (L2, L3). For proper organ development these different tissue layers must interact with each other, though their relative contributions are a matter of debate. Here we use ANGUSTIFOLIA (AN), which controls cell polarity and leaf shape, to study its morphogenetic function in the epidermis and the subepidermis of Arabidopsis thaliana. We show that ANGUSTIFOLIA expression in the subepidermis cannot rescue epidermal cell polarity defects, indicating a cell‐autonomous molecular function. We demonstrate that leaf width is only rescued by subepidermal AN expression, whereas leaf length is also rescued by epidermal expression. Strikingly, subepidermal rescue of leaf width is accompanied by increased cell number in the epidermis, indicating that AN can trigger cell divisions in a non‐autonomous manner.     

Essential Oil of Azorella cryptantha Collected in Two Different Locations from San Juan Province,Argentina: Chemical Variability and Anti‐Insect and Antimicrobial Activities

The essential oils (EOs) of two populations of Azorella cryptantha (Clos) Reiche , a native species from San Juan Province, were obtained by hydrodistillation in a Clevenger‐type apparatus and characterized by GC‐FID and GC/MS analyses. The compounds identified amounted to 92.3 and 88.7% of the total oil composition for A. cryptantha from Bauchaceta (Ac‐BAU) and Agua Negra (Ac‐AN), respectively. The EO composition for the two populations was similar, although with differences in the identity and content of the main compounds and also in the identity of minor components. The main compounds of the Ac‐BAU EO were α‐pinene, α‐thujene, sabinene, δ‐cadinene, δ‐cadinol, trans‐β‐guaiene, and τ‐muurolol, while α‐pinene, α‐thujene, β‐pinene, γ‐cadinene, τ‐cadinol, δ‐cadinene, τ‐muurolol, and a not identified compound were the main constituents of the Ac‐AN EO, which also contained 3.0% of oxygenated monoterpenes. The repellent activity on Triatoma infestans nymphs was 100 and 92% for the Ac‐AN and Ac‐BAU EOs, respectively. Regarding the toxic effects on Ceratitis capitata, the EOs were very active with LD₅₀ values lower than 11 μg/fly. The dermatophytes Microsporum gypseum, Trichophyton rubrum, and T. mentagrophytes and the bacterial strains Escherichia coli LM₁, E. coli LM₂, and Yersinia enterocolitica PI were more sensitive toward the Ac‐AN EO (MIC 125 μg/ml) than toward the Ac‐BAU EO. This is the first report on the composition of A. cryptantha EO and its anti‐insect and antimicrobial properties.     

Ascending auditory interneurons in the cricketTeleogryllus commodus (Walker): comparative physiology and direct connections with afferents

Summary Ascending auditory interneurons of the cricket,Teleogryllus commodus (Walker), were investigated using simultaneous intracellular and extracellular recording in order to identify units which had previously been characterized only by extracellular recording. The morphology and physiology of the large adapting unit (LAU: Fig. 1) and of the small tonic unit (STU: Fig. 2) ofTeleogryllus correspond well to those of the ascending neuron 2 (AN2) and the ascending neuron 1 (AN1) ofGryllus (Figs. 1, 2), respectively.A summary of the ascending auditory interneurons described by various authors in 5 species of crickets is presented in order to establish common identities.Physiological evidence for direct connections between auditory afferents and the ascending auditory interneurons AN1 (STU) and AN2 (LAU) is presented. Simultaneous intracellular recordings from receptors and interneurons in response to sound as well as the activity of auditory interneurons upon electrical stimulation of the tympanal nerve reveal short and constant latencies of receptor-evoked synaptic activity in AN1 (STU) and AN2 (LAU).Abbreviations STU small tonic unit - LAU large adapting unit - AN ascending neuron - EPSP excitatory postsynaptic potential     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015