Anti-echinococcal effect of metformin in advanced experimental cystic echinococcosis: reprogrammed intermediary carbon metabolism in the parasite.

Metformin, a safe biguanide derivative with antiproliferative properties, has shown antiparasitic efficacy against the Echinococcus larval stage. Hence, we assessed the efficacy of a dose of 250 mg kg-1 day-1 in experimental models of advanced CE, at 6 and 12 months post-infection with oral and intraperitoneal administration, respectively. At this high dose, metformin reached intracystic concentrations between 0.7 and 1.7 mM and triggered Eg-TOR inhibition through AMPK activation by AMP-independent and -dependent mechanisms, which are dependent on drug dose. Cystic metformin uptake was controlled by increased expression of organic cation transporters in the presence of the drug. In both experimental models, metformin reduced the weight of parasite cysts, altered the ultrastructural integrity of their germinal layers, and reduced the intracystic availability of glucose, limiting the cellular carbon and energy charge and the proliferative capacity of metacestodes. This glucose depletion in the parasite was associated with a slight increase in cystic uptake of 2-deoxiglucose and the transcriptional induction of GLUT genes in metacestodes. In this context, drastic glycogen consumption led to increased lactate production and altered intermediary metabolism in treated metacestodes. Specifically, the fraction of reducing soluble sugars decreased twofold, and the levels of non-reducing soluble sugars, such as sucrose and trehalose, were modified in both cystic fluid and germinal cells. Taken together, our findings highlight the relevance of metformin as a promising candidate for CE treatment and warrant further research to improve the therapeutic conditions of this chronic zoonosis in humans.

Molecular characterization of a Bacillus thuringiensis strain from Argentina, toxic against Lepidoptera and Coleoptera, based on its whole-genome and Cry protein analysis.

The insecticidal proteins of Bacillus thuringiensis are used in formulations of spore-crystal complexes and their genes have been incorporated into several crops, providing a model for genetic engineering in agriculture. Despite the variability of the Cry proteins described so far, it is still necessary to look for toxins with a broad spectrum of action, since a significant number of pests are not controlled with the available Cry proteins. It is also important to provide alternatives to address the problem of insect resistance, which has already appeared with the use of formulations and with transgenic plants that express cry genes that code for insecticidal proteins. The FCC 7 strain was characterized by the ultrastructural parasporal body under optical and electronic microscopy, and for the detection of Cry8-type proteins by genomic and proteomic approaches. The identity of the strain and the presence of putative toxin encoding genes, and virulence factors analyzed by Illumina Miseq 1500 platform genomic sequencing, was confirmed. The identity of the two Cry8 proteins that make up the parasporal body was confirmed by MALDI-TOF/TOF. To expand knowledge about the insecticidal activity of this strain, we conducted preliminary tests against the cotton boll weevil, Anthonomus grandis. Here we report the characterization of a novel B. thuringiensis isolate native to Argentina (FCC 7) toxic against A. grandis. The strain shows a rounded parasporal body harboring mainly a protein of about 140 kDa and two different types of Cry8 proteins. Through whole-genome sequencing, we identified the presence of two cry8-like crystal protein genes, one vpa-like and two vpb-like genes, and multiple virulence factors, deepening the knowledge of a strain that had already been described as toxic against some lepidopterans and coleopterans, including Spodoptera frugiperda, Anticarsia gemmatalis, Tenebrio molitor and Diabrotica speciosa.

Target of rapamycin signaling is tightly and differently regulated in the plant response under distinct abiotic stresses.

MAIN CONCLUSION: TOR signaling is finely regulated under diverse abiotic stresses and may be required for the plant response with a different time-course depending on the duration and nature of the stress. Target of rapamycin (TOR) signaling is a central regulator of growth and development in eukaryotic organisms. However, its regulation under stress conditions has not yet been elucidated. In Arabidopsis, we show that TOR transcripts and activity in planta are finely regulated within hours after the onset of salt, osmotic, cold and oxidative stress. The expression of genes encoding the partner proteins of the TOR complex, RAPTOR3G and LST8-1, is also regulated. Besides, the data indicate that TOR activity increases at some time during the adverse condition. Interestingly, in oxidative stress, the major TOR activity increment occurred transiently at the early phase of treatment, while in salt, osmotic and cold stress, it was around 1 day after the unfavorable condition was applied. Those results suggest that the TOR signaling has an important role in the plant response to an exposure to stress. Moreover, basal ROS (H2O2) levels and their modification under abiotic stresses were altered in TOR complex mutants. On the other hand, the root phenotypic analysis of the effects caused by the diverse abiotic stresses on TOR complex mutants revealed that they were differently affected, being in some cases less sensitive, than wild-type plants to long-term unfavorable conditions. Therefore, in this work, we demonstrated that TOR signaling is tightly regulated under abiotic stresses, at transcript and activity level, with different and specific time-course patterns according to the type of abiotic stress in Arabidopsis. Taking our results together, we propose that TOR signaling should be necessary during the plant stress response.

Sucrose in bloom-forming cyanobacteria: loss and gain of genes involved in its biosynthesis.

Bloom-forming cyanobacteria are widely distributed in freshwater ecosystems. To cope with salinity fluctuations, cyanobacteria synthesize compatible solutes, such as sucrose, to maintain the intracellular osmotic balance. The screening of cyanobacterial genomes revealed that homologues to sucrose metabolism-related genes only occur in few bloom-forming strains, mostly belonging to Nostocales and Stigonematales orders. Remarkably, among Chroococcales and Oscillatoriales strains, homologues were only found in M. aeruginosa PCC 7806 and Leptolyngbya boryana PCC 6306, suggesting a massive loss of sucrose metabolism in bloom-forming strains of these orders. After a complete functional characterization of sucrose genes in M. aeruginosa PCC 7806, we showed that sucrose metabolism depends on the expression of a gene cluster that defines a transcriptional unit, unique among all sucrose-containing cyanobacteria. It was also demonstrated that the expression of the encoding genes of sucrose-related proteins is stimulated by salt. In view of its ancestral origin in cyanobacteria, the fact that most bloom-forming strains lack sucrose metabolism indicates that the genes involved might have been lost during evolution. However, in a particular strain, like M. aeruginosa PCC 7806, sucrose synthesis genes were probably regained by horizontal gene transfer, which could be hypothesized as a response to salinity fluctuations.

Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats.

Akinetes are resting spore-like cells formed by some heterocyst-forming filamentous cyanobacteria for surviving long periods of unfavourable conditions. We studied the development of akinetes in two model strains of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133, in response to low light and phosphate starvation. The best trigger of akinete differentiation of Anabaena variabilis was low light; that of N. punctiforme was phosphate starvation. Light and electron microscopy revealed that akinetes of both species differed from vegetative cells by their larger size, different cell morphology and large number of intracellular granules. Anabaena variabilis akinetes had a multilayer envelope; those of N. punctiforme had a simpler envelope. During akinete development of Anabaena variabilis, the amount of the storage compounds cyanophycin and glycogen increased transiently, whereas in N. punctiforme, cyanophycin and lipid droplets increased transiently. Photosynthesis and respiration decreased during akinete differentiation in both species, and remained at a low level in mature akinetes. The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles. The results pave the way for genetic and functional studies of akinete differentiation in these species.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees.

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Selection and characterization of Argentine isolates of Trichoderma harzianum for effective biocontrol of Septoria leaf blotch of wheat.

Species of the genus Trichoderma are economically important as biocontrol agents, serving as a potential alternative to chemical control. The applicability of Trichoderma isolates to different ecozones will depend on the behavior of the strains selected from each zone. The present study was undertaken to isolate biocontrol populations of Trichoderma spp. from the Argentine wheat regions and to select and characterize the best strains of Trichoderma harzianum by means of molecular techniques. A total of 84 out of the 240 strains of Trichoderma were able to reduce the disease severity of the leaf blotch of wheat. Thirty-seven strains were selected for the reduction equal to or greater than 50% of the severity, compared with the control. The percentage values of reduction of the pycnidial coverage ranged between 45 and 80%. The same last strains were confirmed as T. harzianum by polymerase chain reaction amplification of internal transcribed spacers, followed by sequencing. Inter-simple sequence repeat was used to examine the genetic variability among isolates. This resulted in a total of 132 bands. Further numerical analysis revealed 19 haplotypes, grouped in three clusters (I, II, III). Shared strains, with different geographical origins and isolated in different years, were observed within each cluster. The origin of the isolates and the genetic group were partially related. All isolates from Paraná were in cluster I, all isolates from Lobería were in cluster II, and all isolates from Pergamino and Santa Fe were in cluster III. Our results suggest that the 37 native strains of T. harzianum are important in biocontrol programs and could be advantageous for the preparation of biopesticides adapted to the agroecological conditions of wheat culture.

Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina.

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent.

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 µM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.

Sucrose synthase in unicellular cyanobacteria and its relationship with salt and hypoxic stress.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, A/UDP-glucose: D: -fructose 2-α-D: -glucosyl transferase) catalyzes a reversible reaction. However, it is in the cleavage of Suc that this enzyme plays an important role in vivo, providing sugar nucleotides for polysaccharide biosynthesis. In cyanobacteria, SuS occurrence has been reported in heterocyst-forming strains, where it was shown to be involved also in nitrogen fixation. We investigated the presence of sequences homologous to SuS-encoding genes (sus) in recently sequenced cyanobacterial genomes. In this work, we show for the first time the presence of SuS in unicellular cyanobacterium strains (Microcystis aeruginosa PCC 7806, Gloebacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1). After functional characterization of SuS encoding genes, we demonstrated an increase in their transcript levels after a salt treatment or hypoxic stress in M. aeruginosa and G. violaceus cells. Based on phylogenetic analysis and on the presence of sus homologs in the most recently radiated cyanobacterium strains, we propose that sus genes in unicellular cyanobacteria may have been acquired through horizontal gene transfer. Taken together, our data indicate that SuS acquisition by cyanobacteria might be related to open up new ecological niches.

Characterization of novel Trichoderma spp. isolates as a search for effective biocontrollers of fungal diseases of economically important crops in Argentina.

Monoconidial cultures of 33 isolates of Trichoderma from Buenos Aires Province, Argentina were characterized on the basis of twenty eight morphological, physiological and biochemical features. All of them were screened for proteinase, endochitinase and ß-1,3 glucanase activity. Universally primed PCR (UP-PCR) and inter-simple sequence repeat (ISSR) techniques were used to examine the genetic variability among isolates, which resulted in 127 bands for the total number of isolates. These results were subjected to numerical analysis revealing 20 haplotypes grouped in five clusters. The ability of Trichoderma isolates to antogonize soil-borne fungal plant pathogens using a dual culture assay was done against five fungal species: Alternaria sp., Bipolaris sorokiniana, Fusarium graminearum, F. solani, and Pyricularia oryzae. The highest inhibition values (85% RI) were obtained against B. sorokiniana and P. oryzae. Three isolates of T. harzianum named as FCCT2, FCCT3 and FCCT9 were capable of causing a high growth inhibition on four of the fungal species assayed, which was in agreement with their higher extracellular hydrolytic activity. Our results suggest that these isolates have the potential to be effective agents for biocontrol of cereal and tomato fungal pathogens.

A Promising Use of Trimethyl Chitosan for Removing Microcystis aeruginosa in Water Treatment Processes.

The increase in cyanobacterial blooms linked to climate change and the eutrophication of water bodies is a global concern. The harmful cyanobacterium Microcystis aeruginosa is one of the most common bloom-forming species whose removal from fresh water and, in particular, from that used for water treatment processes, remains a crucial goal. Different biodegradable and environmentally friendly coagulants/flocculants have been assayed, with chitosan showing a very good performance. However, chitosan in its original form is of limited applicability since it is only soluble in acid solution. The objective of this work was therefore to test the coagulant/flocculant capacity of trimethylchitosan (TMC), a chitosan derivative produced from residues of the fishing industry. TMC has a constitutively net positive charge enabling it to remain in solution regardless of the pH. Results show that even at alkaline pHs, common during cyanobacterial blooms, TMC is effective in removing buoyant cyanobacteria from the water column, both in test tube and Jar-Test experiments. Cell integrity was confirmed by fluorescent stain and electron microscopy. Our findings lead us to conclude that the use of TMC to remove bloom cells early in the treatment of drinking water is both feasible and promising.

TOR signaling in the green picoalga Ostreococcus tauri.

Target of rapamycin (TOR) is a master regulator that controls growth and metabolism by integrating external and internal signals. Although there was a great progress in the study of TOR in plants and in the model alga Chlamydomonas, scarce data are available in other green algae. Thus, in this work we studied TOR signaling in Ostreococcus tauri, the smallest free-living eukaryote described to date. This picoalga is particularly important because it has a key site at the base of the green lineage and is part of the marine phytoplankton, contributing to global photosynthesis. We investigated OtTOR complex in silico and experimentally, by using first- and second-generation TOR inhibitors, such as rapamycin and PP242. We analyzed the effect of TOR down-regulation on cell growth and on the accumulation of carbon reserves. The results showed that O. tauri responds to TOR inhibitors more similarly to plants than to Chlamydomonas, being PP242 a valuable tool to study this pathway. Besides, Ottor expression analysis revealed that the kinase is dynamically regulated under nutritional stress. Our data indicate that TOR signaling is conserved in O. tauri and we propose this alga as a good and simple model for studying TOR kinase and its regulation.

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria.

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.

Differential roles of alkaline/neutral invertases in Nostoc sp. PCC 7120: Inv-B isoform is essential for diazotrophic growth.

The presence of two alkaline/neutral invertases (Inv-A and Inv-B) in the filaments of Nostoc (also named Anabaena) sp. strain PCC 7120 and the involvement of sucrose metabolism in nitrogen fixation led us to investigate the physiological function of those isoforms in cells growing under different nitrogen sources. The highest expression level of each encoding gene was obtained in the presence of ammonium. These results were paralleled by polypeptide and enzyme activity level. In cells of N(2)-fixing filaments, localization of gene expression and subcellular enzyme activity assays demonstrated that invA gene (alr1521) expresses only in vegetative cells, whereas for invB (alr0819), expression is detected in both vegetative cells and heterocysts. In contrast to invA, when invB was knocked out, the filaments were unable to grow on diazotrophic conditions and the accumulation of sucrose and glycogen was altered. Our results demonstrate an essential role for Inv-B for diazotrophic growth and that Inv-B plays a key role in the coordination of sucrose and glycogen metabolism. We can also suggest that invB is likely to integrate the repertoire of genes regulated by a cyanobacterial transcription factor (NtcA) that plays a central role in global nitrogen control.

Genetic diversity of cry gene sequences of Bacillus thuringiensis strains analyzed by denaturing gradient gel electrophoresis.

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.

Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803.

Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 µM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803.

Protein phosphatase activity and sucrose-mediated induction of fructan synthesis in wheat.

In this work, we analyze protein phosphatase (PP) involvement in the sucrose-mediated induction of fructan metabolism in wheat (Triticum aestivum). The addition of okadaic acid (OA), a PP-inhibitor, to sucrose-fed leaves reduced fructosylsucrose-synthesizing activity (FSS) induction in a dose-dependent manner. The expression of the two enzymes that contribute to FSS activity, 1-SST (1-sucrose:sucrose fructosyltransferase, E.C. 2.4.1.99) and 6-SFT (6-sucrose:fructan fructosyltransferase, E.C. 2.4.1.10), was blocked by 1 microM OA. These results suggest the involvement of a PP type 2A in sucrose signaling leading to fructan synthesis. OA addition to the feeding medium impaired both sucrose accumulation in leaves and the expression of sucrose-H+ symporter (SUT1). It is known that sucrose concentration must exceed a threshold for the induction of fructan metabolism; hence PP2A inhibition may result in lower sucrose levels than required for this induction. OA also induced the vacuolar acid invertase (acid INV) transcript levels suggesting that PP activity might play a role in carbon partitioning. Total extractable PP2A activity decreased during 24 h of treatment with sucrose, in parallel with declining sugar uptake into leaf tissues. In conclusion, our results suggest that PP2A is involved in sucrose-induction of fructan metabolism and may play a role in regulating sucrose uptake, but do not rule out that further steps in sucrose signaling pathway may be affected.

Role of NtcA, a cyanobacterial global nitrogen regulator, in the regulation of sucrose metabolism gene expression in Anabaena sp. PCC 7120.

In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.