Effects of biochar-immobilized bacteria on phytoremediation of cadmium-polluted soil.

This work is the first report of the ability of biochar-immobilized cadmium-resistant bacteria (CRB) on promoting the efficiency of cadmium phytoextraction by Chlorophytum laxum R.Br. The survival of CRB immobilized on biochar in cadmium-contaminated soil at a concentration of 75.45 mg kg-1 was studied. The results found that both CRB, namely Arthrobacter sp. TM6 and Micrococcus sp. MU1, can survive and grow in cadmium-contaminated soil. To study phytoextraction in the pot experiments, 2-month-old C. laxum was individually planted in cadmium-contaminated soil and divided into four treatments, including (i) untreated control, (ii) biochar, (iii) biochar-immobilized (BC) Arthrobacter sp., and (iv) BC-Micrococcus sp. The results found that biochar-immobilized CRB did not cause any effect to the root lengths and shoot heights of plants compared to the untreated control. Interestingly, inoculation of biochar-immobilized CRB significantly increased cadmium accumulation in the shoots and roots compared to the untreated control. In addition, the highest cadmium content in a whole plant, best phytoextraction performance, and greatest bioaccumulation factor was found in plant inoculated with BC-Micrococcus sp., followed by BC-Arthrobacter sp. In conclusion, inoculation of biochar-immobilized CRB enhanced cadmium accumulation and translocation of cadmium from the roots to shoots, suggesting further applying biochar-immobilized CRB in cadmium-polluted soil for promoting cadmium phytoextraction efficiency of ornamental plants. Graphical abstract.

Cadmium-affected synthesis of exopolysaccharides by rhizosphere bacteria.

AIM: Study is focused on the influence of cadmium addition to growth media on production yield, their size and molecular mass of exopolysaccharides (EPS) synthesized by three rhizosphere bacteria strains. Inhibition of bacterial growth by increasing concentrations of Cd2+ was also analysed. METHODS AND RESULTS: The highest impact of Cd2+ was noticed on the growth of Arthrobacter sp. and Rhizobium metallidurans. Chryseobacterium sp. and Arthrobacter sp. produced significantly lower when compared to R. metallidurans amounts of EPS under the influence of Cd2+ . In all bacterial strains both size and molecular mass decreased after addition of Cd2+ to growth media. It causes a change in EPS conformation to more planar, which minimizes the volume of liquid in the interglobular space next to the bacterial wall. Results confirmed strong effect of Cd2+ on the structure and synthesis of bacterial EPS what can be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal polluted soils. CONCLUSION: This work proves that due to the presence of cadmium ions, the size and conformation of EPS produced by selected bacterial strains is changed to minimize their impact on cell. We suggest that shifting in EPS conformation from bigger globular particles to the smaller planar ones could be one of the probable mechanisms of Cd resistance in metallotolerant bacteria, and finally explain increased efficiency of heavy metal phytoextraction by EPS-producing plant growth-promoting micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the most promising remediation technique for Cd-contaminated areas is the phytoremediation in which rhizosphere bacteria play an important role by protecting plants' roots from toxic condition thus enhancing efficiency of intake. EPS secretion by bacteria is one of the most common mechanisms to protect the cell from impact of unpleasant environmental conditions, for example, toxicity of heavy metals like Cd.

Comparative transcriptomic and proteomic analysis of Arthrobacter sp. CGMCC 3584 responding to dissolved oxygen for cAMP production.

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

Phytoremediation of cadmium-polluted soil by Chlorophytum laxum combined with chitosan-immobilized cadmium-resistant bacteria.

This study examined the performance of the chitosan-immobilized cadmium-resistant bacteria Arthrobacter sp. and Micrococcus sp. on cadmium phytoremediation by Chlorophytum laxum in cadmium-polluted soil. These immobilized cadmium-resistant bacteria can survive in cadmium-contaminated soil and significantly increased soil cadmium solubility, but the ability of chitosan-immobilized cells to increase cadmium solubility was lower than that of free cells. A pot experiment demonstrated that chitosan-immobilized Micrococcus sp. promoted the growth of C. laxum planted in cadmium-contaminated soil. A significant increase in the cadmium concentration in the roots and aboveground parts of C. laxum was found in plants inoculated with free and chitosan-immobilized cells of these bacteria. The performance of Arthrobacter sp. free cells to augment cadmium accumulation in C. laxum was a little bit better than that of chitosan-immobilized Arthrobacter sp., except at 9 weeks after planting. The phytoextraction coefficient, bioaccumulation factor, and translocation factor of C. laxum inoculated with free and chitosan-immobilized cells of cadmium-resistant bacteria were higher than those of the uninoculated control and increased with time. Our findings suggest that chitosan-immobilized cells can be exploited to enhance the efficiency of cadmium phytoremediation by C. laxum.

RfiA, a novel PAP2 domain-containing polytopic membrane protein that confers resistance to the FtsZ inhibitor PC190723.

BACKGROUND: As an essential protein for bacterial cell division, the tubulin-like FtsZ protein has been selected as a target for development of next generation antimicrobials. PC190723 is a fluoride-containing benzamide compound developed as a FtsZ inhibitor that selectively inhibits growth of multidrug resistant Gram-positive bacteria. AIM: Our aim was to investigate the mechanism of resistance to PC109723 conferred by over-expression of a gene, rfiA, in an environmental bacterium Arthrobacter A3. MATERIALS & METHODS: The investigations included analysis of the effect of PC109723 on wild-type Arthrobacter A3 and a recombinant strain over-expressing rfiA, in vivo localization of RfiA, in vitro measurements of fluorine release from PC109723 by membrane extracts from the over-expression strain combined with mass spectrophotometric analysis of reaction products, and modelling of RfiA structure. RESULTS: We describe a novel protein, RfiA, from Arthrobacter A3 that confers PC190723 resistance. RfiA is a PAP2 domain-containing polytopic transmembrane protein that can modify the fluoridated benzamide ring that is critical for high affinity binding of PC190723 with FtsZ. CONCLUSION: RfiA-mediated modification of PC190723 is the first reported instance of resistance to this antibiotic involving a change to its structure. We predict that adoption of PC190723 or related benzamides as antimicrobials in clinical practice will lead to the acquisition by resistant pathogens of a gene encoding this subfamily of proteins.

Tutorial for writing systematic reviews for the Brazilian Journal of Physical Therapy (BJPT) / Tutorial para elaboração de revisões sistemáticas para o Brazilian Journal of Physical Therapy (BJPT)

Systematic reviews aim to summarize all evidence using very rigorous methods in order to address a specific research question with less bias as possible. Systematic reviews are widely used in the field of physical therapy, however not all reviews have good quality. This tutorial aims to guide authors of the Brazilian Journal of Physical Therapy on how systematic reviews should be conducted and reported in order to be accepted for publication. It is expected that this tutorial will help authors of systematic reviews as well as journal editors and reviewers on how to conduct, report, critically appraise and interpret this type of study design. .

Revisões sistemáticas têm como objetivo sumarizar toda a evidência disponível, através de métodos rigorosos, para responder a uma pergunta de pesquisa específica com o mínimo de viés possível. Revisões sistemáticas são amplamente utilizadas na fisioterapia, porém nem todas as revisões possuem boa qualidade. Esse tutorial tem como objetivo guiar os autores do Brazilian Journal of Physical Therapy sobre como revisões sistemáticas deveriam ser conduzidas e descritas para que sejam aceitas para publicação. Espera-se que esse tutorial irá auxiliar autores de revisões sistemáticas, assim como editores e revisores de periódicos em como conduzir, descrever, fazer análise crítica e interpretar esse tipo de delineamento de pesquisa.

Chemical composition and antibacterial activity of essential oils of Lantana camara, Ageratum houstonianum and Eupatorium adenophorum.

Essential oils have applications in folk medicine, food preservation, and as feed additives. The essential oils of Lantana camara Linn. (Verbenaceae), Ageratum houstonianum Mill. (Asteraceae) and Eupatorium adenophorum Spreng. (Asteraceae) were analyzed by Gas chromatography-mass spectrometry (GCMS). In L. camara oil, of the total identified (83.91%) volatile constituents, five constituents [3,7,11-trimethyl-1,6,10-dodecatriene (28.86%), beta-caryophyllene (12.28%), zingiberene (7.63%), gamma-curcumene (7.50%) and alpha-humulene (3.99%)] represented the major ones. In A. houstonianum oil, among the total identified volatile constituents (94.51%), three [precocene-II (52.64%), precocene-I (22.45%) and beta-caryophyllene (9.66%)] represented the major ones. In E. adenophorum oil, of the total identified volatile constituents (84.95%), six [1-napthalenol (17.50%), alpha-bisabolol (9.53%), bornyl acetate (8.98%), beta-bisabolene (6.16%), germacrene-D (5.74%) and alpha- phellandrene (3.85%)] represented the major ones. The antibacterial activity expressed as Minimum Bactericidal Concentration (MBC) (microg/mL) was determined by the broth dilution method. The essential oil of E. adenophorum had antibacterial activity against Arthrobacter protophormiae, Escherichia coli, Micrococcus luteus, Rhodococcus rhodochrous, and Staphylococcus aureus with MBC values of 200, 100, 100, 12.5, and 200, respectively. The essential oil of A. houstonianum showed antibacterial activity against M. luteus and R. rhodochrous with MBC of 100 and 12.5, but not against A. protophormiae, E. coli, and S. aureus. The essential oil of L. camara showed antibacterial activity against A. protophormiae, M. luteus, R. rhodochrous and S. aureus with MBC of 50, 25, 12.5, and 200, respectively, but not against E. coli. MBC was lowest for R. rhodochrous for all the three essential oils.

Enhanced cyclic adenosine monophosphate production by Arthrobacter A302 through rational redistribution of metabolic flux.

Cyclic adenosine monophosphate (cAMP) was synthesized through the purine salvage synthesis pathway by Arthrobacter A302. Results showed that hypoxanthine was the best of the precursors, and the cAMP concentration reached 4.06 g/L. For inhibition of the glycolytic pathway, sodium fluoride was found the optimal effector, which was further studied on cAMP production. With the addition of 0.4 g/L of sodium fluoride, the maximal cAMP concentration reached 11.04 g/L, and the concentrations of lactic acid, alpha-ketoglutarate and citric acid were decreased by 77%, 86% and 76%, respectively. Meanwhile, the specific activities of glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase were decreased by 66%, 61%, and 46%, respectively. By contrast the activity of 6-phosphoglucose dehydrogenase was increased by 100%, which demonstrated the redistribution of metabolic flux. This is the first study to reveal the regulatory mechanisms of different effectors on cAMP production among the EMP pathway, HMP pathway and TCA cycle.

Isolation and characterization of four gram-positive nickel-tolerant microorganisms from contaminated sediments.

Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.

A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans.

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.

[Effect of quinocitrinines from the fungus Penicillium citrinum on the respiration of yeasts and bacteria].

We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.

Horizontal gene transfer of PIB-type ATPases among bacteria isolated from radionuclide- and metal-contaminated subsurface soils.

Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with co-occurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb(r)) isolates was amplified with PCR primers specific for P(IB)-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P(IB)-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb(r) P(IB)-type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO(2)(2+)) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P(IB)-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.

[Employment of associative bacteria for the inoculation of barley plants cultivated in soil contaminated with lead and cadmium]. / Ispol'zovanie assotsiativnykh bakterii dlia inokuliatsii iachmenia v usloviiakh zagriazneniia pochvy svintsom i kadmiem.

In laboratory experiments, the rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10, and Flavobacterium sp. L30 were found to have a relatively high resistance to the toxic heavy metals lead and cadmium (except that strain L30 was found to be sensitive to Cd). When introduced by means of seed bacterization, the heavy metal-resistant strains actively colonized the rhizosphere of barley plants cultivated in uncontaminated and contaminated soils. In both pot and field experiments, seed bacterization improved the growth of barley plants and the uptake of nutrient elements from soil contaminated with Pb and Cd. The bacterization also prevented the accumulation of Pb and Cd in barley plants, thereby mitigating the toxic effect of these heavy metals on the plants.

Isolation of ALU1-P gene encoding a protein with aluminum tolerance activity from Arthrobacter viscosus.

We isolated a DNA fragment (ALU1-P) encoding a protein with an activity of aluminum tolerance from an Al tolerant soil microorganism, Arthrobacter viscosus. This microorganism was isolated from acidic tea field soils. The cloned DNA is composed of 1090 nucleotides, which has one open-reading frame without any stop codon. However, when the DNA fragment was transferred into Escherichia coli, a microorganism susceptible to Al toxicity, it endowed E. coli with Al tolerance. The deduced amino acid sequence of the DNA showed 65% identity with the protein of YbaX gene in Escherichia coli, and 51.1% identity with YB91 Haein hypothetical protein of HI1191 gene in Haemophilus influenzae. The ALU1-P gene in the expression vector produced a protein of 192 amino acids deriving a molecular weight of 21.3 kDa by using the stop codon in vector. The ALU1-P gene is a new one that has the characteristic of Al tolerance.

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis.

Aluminum (0.74 mM) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with delta-aminolevulinic acid (delta-ALA, 1.2 mM) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. delta-ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 +/- 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.

Vermiculin derivatives. Part 1: Synthesis of vermiculin derivatives.

Hydrogenation of the macrodiolide antibiotic vermiculin (1) over Adams catalyst afforded [8S, 16S]-8,16-bis(2'-oxopropyl)-1,9- dioxyacyclohexadeca-2,5,10,13-tetrone (2), [8S, 16S]-8,16-bis(2'-oxopropyl)-13-hydroxy-1,9-dioxacyclohexadeca- 2,5,10-trione (3), [8S,16S]-8,16-bis(2' oxopropyl)-1,9- dioxacyclohexadeca-2,5,10-trione (4) together with [7S]-4,9-dioxo-7-(4',9'-dioxodecanoyloxy)decanoic acid (5). Hydrogenation of diolide 1 over Pd/C gave tetrahydrovermiculin (2) only. The prepared compounds showed lower antibacterial and cytotoxic activities than vermiculin (1).

L-lysine production by S-2-aminoethyl-L-cysteine-resistant mutants of Arthrobacter globiformis.

Using mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a number of homoserine auxotrophs have been isolated from a glutamate-producing Arthrobacter globiformis excreting L-lysine in good amounts. For further improvement, mutants resistant to the lysine analog S-(2-aminoethyl)-L-cysteine have been isolated from homoserine auxotrophs. For the three potent mutants tested, White's medium was found to be the best. Glucose, ammonium nitrate and biotin were found to be optimum at 280 mmol/L, 40 mmol/L and 22 nmol/L, respectively. With optimal glucose, ammonium nitrate and biotin, the strain AECrVI yielded 36 g lysine per L in flask culture.

Production and isolation of siderophores from the soil fungus Epicoccum purpurascens.

A large number of iron transport agents, siderophores, which stimulated the growth of Arthrobacter flavescens JG-9, were isolated during a study of the antitumor activity associated with the metabolic products of the fungus Epicoccum purpurascens. The production of the siderophores was significantly enhanced in a variety of media by culture of the fungus in the near absence of ferric iron. A novel method of purification involving a carboxylic ion-exchange resin separated the siderophores into four subgroups. The first subgroup, which contained the majority of the activity, was subsequently resolved in a similar manner with the carboxylic resin into seven individual siderophores. Of these, two were characterized as ferricrocin and coprogen whereas the others appeared to represent new compounds. One of the latter was given the name triornicin and exhibited slight antitumor activity in mice injected with Ehrlich ascites tumor cells.

Photodynamic induction of a bacterial cell surface polypeptide.

The photodynamic action of several dyes on cells of a bacterium, tentatively identified as a species of Arthrobacter, resulted in remarkable stimulation of synthesis of a polypeptide 21,000 daltons in mass. This polypeptide resides on the cell surface and can be solubilized by sodium dodecyl sulfate without lysis of the cells. Chlorophyllin and rose bengal are effective in inducing synthesis of the polypeptide in proportion to their ability to sensitize the photooxidation of histidine. Etiolated cells of the alga Chlamydomonas reinhardtii y-1 excrete a substance into the medium that also sensitized the photoinduction of the polypeptide.

L-serine dehydratase from Arthrobacter globiformis.

1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.