The Bacillus subtilis YkuV is a thiol:disulfide oxidoreductase revealed by its redox structures and activity

The Bacillus subtilis YkuV responds to environmental oxidative stress and plays an important role for the bacteria to adapt to the environment. Bioinformatic analysis suggests that YkuV is a homolog of membrane-anchored proteins and belongs to the thioredoxin-like protein superfamily containing the typical Cys-Xaa-Xaa-Cys active motif. However, the biological function of this protein remains unknown thus far. In order to elucidate the biological function, we have determined the solution structures of both the oxidized and reduced forms of B. subtilis YkuV by NMR spectroscopy and performed biochemical studies. Our results demonstrated that the reduced YkuV has a low midpoint redox potential, allowing it to reduce a variety of protein substrates. The overall structures of both oxidized and reduced forms are similar, with a typical thioredoxin-like fold. However, significant conformational changes in the Cys-Xaa-Xaa-Cys active motif of the tertiary structures are observed between the two forms. In addition, the backbone dynamics provide further insights in understanding the strong redox potential of the reduced YkuV. Furthermore, we demonstrated that YkuV is able to reduce different protein substrates in vitro. Together, our results clearly established that YkuV may function as a general thiol:disulfide oxidoreductase, which acts as an alternative for thioredoxin or thioredoxin reductase to maintain the reducing environment in the cell cytoplasm.     

DsbL and DsbI form a specific dithiol oxidase system for periplasmic arylsulfate sulfotransferase in uropathogenic Escherichia coli

Disulfide bond formation in the Escherichia coli periplasm requires the transfer of electrons from substrate proteins to DsbA, which is recycled as an oxidant by the membrane protein DsbB. The highly virulent, uropathogenic E. coli strain CFT073 contains a second, homologous pair of proteins, DsbL and DsbI, which are encoded in a tri-cistronic operon together with a periplasmic, uropathogen-specific arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI form a functional redox pair, and that ASST is a substrate of DsbL/DsbI in vivo. DsbL is the most reactive oxidizing thioredoxin-like protein known to date. In contrast to DsbA, however, DsbL oxidizes reduced RNaseA with a much lower rate and prevents unspecific aggregation of reduced insulin. The 1.55 Å resolution crystal structure of reduced DsbL provides insight into the reduced state of thioredoxin-like dithiol oxidases at high resolution, and reveals an unusual cluster of basic residues stabilizing the thiolate anion of the nucleophilic active-site cysteine. We propose that the DsbL/DsbI pair of uropathogenic E. coli was acquired as an additional, specific redox couple that guarantees biological activity of ASST.     

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.     

Solution structure of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins

Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13-->Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins.     

Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here.HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation.Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor.MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues.ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors.ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H₂O₂. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.     

Enhancement of auranofin-induced lung cancer cell apoptosis by selenocystine,a natural inhibitor of TrxR1 in vitro and in vivo

Thioredoxin reductase (TrxR) is overpressed in many human tumors and has a key role in regulating intracellular redox balance. Recently, thioredoxin system has emerged as a valuable target for anticancer drug development. Herein we demonstrate that selenocystine (SeC) could enhance auranofin (AF)-induced A549 human lung adenocarcinoma cell apoptosis in vitro and in vivo through synergetic inhibition of TrxR1. SeC pretreatment significantly enhanced AF-induced loss of mitochondrial membrane potential (Δψ_m) by regulating Bcl-2 family proteins. The combined treatment with SeC and AF also resulted in enhanced intracellular reactive oxygen species (ROS) accumulation, DNA damage, and inactivation of ERK and AKT. Inhibitors of ERK and AKT effectively enhanced combined treatment-induced apoptotic cell death. However, inhibition of ROS reversed the apoptosis induced by SeC and AF, and recovered the inactivation of ERK and AKT, which revealed the importance of ROS in cell apoptosis and regulation of ERK and AKT pathways. Moreover, xenograft lung tumor growth in nude mice was more effectively inhibited by combined treatment with SeC and AF by induction of apoptosis through targeting TrxR1 in vivo. Taken together, our results suggest the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism by targeting TrxR1.     

Recruitment of thioredoxin-like domains into prostaglandin synthases

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently. We have identified two new enzymes, thioredoxin-type PGF synthase and membrane-associated PGE synthase-2, with thioredoxin-like domains. In addition, the N-terminal domain of hematopoietic PGD synthase is classified into the thioredoxin-like superfamily, based on structural similarity. The active sites of the former two enzymes have a CXXC motif, which is also critical for the thioredoxin activity. In contrast, hematopoietic PGD synthase lacks the motif, and the activity is carried out by glutathione. A phylogenetic tree of the thioredoxin-like domains suggests that they have been independently recruited into these PG synthases. We will discuss the functional meaning of the thioredoxin-like domains in the PG synthases from the viewpoint of the redox activity.     

Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif

The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247–D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the ‘thioredoxin-like clan’. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.     

Serum CXCL5 level is associated with tumor progression in penile cancer

Chemokine (C-X-C motif) ligand 5 is an important regulator of tumor progression in many cancers, and could serve as potential serum cancer biomarker. Our initial analysis identified CXCL5 as a cancer-related gene highly expressed in PC. Patients with PC exhibited markedly higher preoperative serum CXCL5 levels compared with that in healthy individuals (P<0.001). The area under the curve (AUC) was 0.880 with the sensitivity of 84.0%, and specificity of 80.4% to distinguish PC. Serum CXCL5 levels were also significantly decreased following tumor resection in patients with PC (P=0.001). Preoperative serum CXCL5 level was significantly associated with clinicopathological characteristics including T stage (P=0.001), nodal status (P<0.001), and pelvic lymph node metastasis (P=0.018). Cox regression analysis showed that serum CXCL5 level could serve as an independent prognostic factor for disease-free survival with a HR of 6.363 (95% CI: 2.185–18.531, P=0.001). CXCL5 and its receptor CXCR2 exhibited correlated expression pattern in PC tissues. Differential CXCL5 expression was observed in normal penile tissues, PC cell lines, and their culture supernatants. Furthermore, knockdown of CXCL5 or CXCR2 expression markedly suppressed malignant phenotypes (cell proliferation, clonogenesis, apoptosis escape, migration, and invasion), attenuated STAT3 and AKT signaling, and reduced MMP2/9 secretion in PC cell lines. In conclusion, our findings revealed that serum CXCL5 level might serve as a potential diagnostic and prognostic cancer biomarker for penile cancer. Autocrine CXCL5/CXCR2 signaling might activate multiple downstream oncogenic signaling pathways (STAT3, AKT, MMP2/9) to promote malignant progression of PC, which may warrant further investigation in the future.     

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Identification of the three-dimensional thioredoxin motif: related structure in the ORF3 protein of the Staphylococcus aureus mer operon.

We have developed a computerized search pattern for recognition of the three-dimensional redox site of thioredoxins based on primary and predicted secondary structure. This pattern, developed in the ARIADNE protein expert system, is used to search for thioredoxin-like tertiary structural motif among proteins for which the only structural information is the primary sequence. The pattern was trained on 102 protein sequences (25 functionals and 77 controls); it matches all 25 members of the functional set under cutoff conditions that include only 2 members of the control set, for a sensitivity of 1.0 and a specificity of 0.97. The pattern matches only one of the two thioredoxin-like domains in protein disulfide isomerases (PDIs) and their analogues, suggesting that the C-terminal domain is more structurally similar to thioredoxin than the N-terminal domain. The Escherichia coli DsbA protein, a possible PDI analogue, appears to be more structurally similar to the N-terminal thioredoxin-like domain of PDIs. Thioredoxin-like redox functionality has been proposed for lutropin and follitropin, in part on the basis of their having -Cys-X-Pro-Cys- sequences. None match our pattern; all lack a predicted alpha-helix pattern element immediately after the active site. Hypothetical proteins in the National Biomedical Research Foundation Protein Identification Resource database were searched for matches to the pattern. The most interesting match was a hypothetical protein (161 residues) from the third open reading frame in the Staphylococcus aureus mer operon, which is involved in mercury detoxification. The match to our pattern and the hydrophobicity distribution in aligned elements of secondary structure not in our pattern strongly suggest that it has thioredoxin-like structure.     

The thioredoxin homolog YbbN functions as a chaperone rather than as an oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20 kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor,ARQ 092

As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.     

Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.     

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

Background

The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively.

Results

We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions.

Conclusion

P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.