Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein.

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.

Crystal Structures of Plk1 Polo-Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2-Derived Peptide.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.

Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1.

Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak-Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1.

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Structural and biochemical analysis of the PTPN4 PDZ domain bound to the C-terminal tail of the human papillomavirus E6 oncoprotein.

High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes.

Signaling Pathways Regulated by UBR Box-Containing E3 Ligases.

UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases.

Development of an analytical method for multi-residue quantification of 18 anthelmintics in various animal-based food products using liquid chromatography-tandem mass spectrometry.

In this study, we developed a simple screening procedure for the determination of 18 anthelmintics (including benzimidazoles, macrocyclic lactones, salicylanilides, substituted phenols, tetrahydropyrimidines, and imidazothiazoles) in five animal-derived food matrices (chicken muscle, pork, beef, milk, and egg) using liquid chromatography-tandem mass spectrometry. Analytes were extracted using acetonitrile/1% acetic acid (milk and egg) and acetonitrile/1% acetic acid with 0.5 mL of distilled water (chicken muscle, pork, and beef), and purified using saturated n-hexane/acetonitrile. A reversed-phase analytical column and a mobile phase consisting of (A) 10 mM ammonium formate in distilled water and (B) methanol were used to achieve optimal chromatographic separation. Matrix-matched standard calibration curves (R 2 ≥0.9752) were obtained for concentration equivalent to ×1/2, ×1, ×2, ×3, ×4, and ×5 fold the maximum residue limit (MRL) stipulated by the Korean Ministry of Food and Drug Safety. Recoveries of 61.2-118.4%, with relative standard deviations (RSDs) of ≤19.9% (intraday and interday), were obtained for each sample at three spiking concentrations (×1/2, ×1, and ×2 the MRL values). Limits of detection, limits of quantification, and matrix effects were 0.02-5.5 µg/kg, 0.06-10 µg/kg, and -98.8 to 13.9% (at 20 µg/kg), respectively. In five samples of each food matrix (chicken muscle, pork, beef, milk, and egg) purchased from large retailers in Seoul that were tested, none of the target analytes were detected. It has therefore been shown that this protocol is adaptable, accurate, and precise for the quantification of anthelmintic residues in foods of animal origin.

R-catcher, a potent molecular tool to unveil the arginylome.

Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18.

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.

Simultaneous Quantification of Chloramphenicol, Thiamphenicol, Florfenicol, and Florfenicol Amine in Animal and Aquaculture Products Using Liquid Chromatography-Tandem Mass Spectrometry.

The accumulation of antimicrobial residues in edible animal products and aquaculture products could pose health concerns to unsuspecting consumers. Hence, this study aimed to develop a validated method for simultaneous quantification of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in beef, pork, chicken, shrimp, eel, and flatfish using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary-secondary amine (PSA) and MgSO4 were used for sample purification. The analytes were separated on a reversed-phase analytical column. The coefficients of determination for the linear matrix-matched calibration curves were ≥0.9941. Recovery rates ranged between 64.26 and 116.51% for the four analytes with relative standard deviations (RSDs) ≤ 18.05%. The calculated limits of detection (LODs) and limits of quantification (LOQs) were 0.005-3.1 and 0.02-10.4 µg/kg, respectively. The developed method was successfully applied for monitoring samples obtained from local markets in Seoul, Republic of Korea. The target residues were not detected in any tested matrix. The designed method was versatile, sensitive, and proved suitable for quantifying residues in animal-derived products.

Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1-RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

Determination of endrin and δ-keto endrin in five food products of animal origin using GC-µECD: A modified QuEChERS approach to traditional detection.

A simple quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based method was developed for the analysis of endrin and its metabolite, δ-keto endrin, in five animal-derived food products (chicken, pork, beef, egg, and milk) using a gas chromatography-micro electron capture detector (GC-µECD). Samples were extracted with acidified acetonitrile, salted out with magnesium sulfate and sodium acetate, and finally purified with a dual layer solid-phase extraction cartridge (SPE) that contains both Supelclean ENVI-Carb (upper layer) and primary secondary amine (lower layer) SPE sorbents. A seven-point external calibration curve was constructed both for the solvent and matrix for both compounds. Good linearity was achieved for both analytes, with coefficients of determination (R2) ≥ 0.9960. The limits of detection (LODs) were 0.003 mg/kg, whereas the limits of quantification (LOQ) were 0.01 mg/kg, which were 10 times lower than the extraneous maximum residue limit (EMRL) designated by CODEX Alimentarius for the specified matrices. The method was validated via recovery performances in triplicates, with three fortification levels equivalent to LOQ, 2 × LOQ, and 10 × LOQ. The method provided excellent recoveries, ranging between 75.63 and 117.92%, with relative standard deviations (RSD) ≤ 8.52% for both analytes in various matrices. The developed method was successfully applied to monitor market samples collected from 20 different places throughout the Republic of Korea, and none of the tested analytes were found in the analyzed samples. Conclusively, we could propose that the current method can be used for routine analysis of endrin and δ-keto endrin in any type of fatty food matrix.

Method development, matrix effect, and risk assessment of 49 multiclass pesticides in kiwifruit using liquid chromatography coupled to tandem mass spectrometry.

In the present study, a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method with a minimal matrix effect (ME) was developed and validated for simultaneous determination of a diverse range of pesticides (49) in kiwifruit. Samples extracted by the quick, easy, cheap, effective, rugged, and safe (QuEChERS) citrate-buffered method were analyzed either without purification or following purification (with primary secondary amine (PSA) or PSA + graphitized carbon black (GCB)). With the addition of a clean-up step, the suppression of the ME decreased, with a higher number of pesticides determined by the application of PSA + GCB. The method exhibited good linearity with coefficients of determination (R2) ≥ 0.9972 and satisfactory recoveries (70-120%) with a relative standard deviations (RSDs) <10%. The limits of quantification (LOQ) were lower than the maximum residue limits (MRLs) set by the Korean Ministry of Food and Drug Safety (MFDS) and the CODEX Alimentarius. The developed method was applied to the real samples and the results indicated that the quantitated levels of all pesticides, except for pyraclostrobin and carbendazim, are lower than the MRLs set by the regulatory authorities. The percentage of the acceptable daily intake was <20%, suggesting that there is no risk associated with the intake of residual pesticides through kiwifruit.

A quick and effective methodology for analyzing dinotefuran and its highly polar metabolites in plum using liquid chromatography-tandem mass spectrometry.

A simple and effective method was developed for analyzing dinotefuran and its three metabolites (MNG, UF, and DN) in plum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Due to the polarity and high water miscibility, dinotefuran and some of its metabolites (especially DN) have some limitations to be extracted with acetonitrile and salt following the "QuEChERS" sample preparation methodology. Alternatively, the samples were extracted with methanol, and purified with dispersive-solid phase extraction procedure (d-SPE) using primary secondary amine (PSA) and C18 sorbents after filtration, and mass up. Due to the suppression effect originated from plum matrix, matrix-matched calibration curves, which provided good linearity with coefficient of determination (R2)≥0.998, were used for quantification of all analytes. Blank plum samples fortified with 2 spiking levels (10×LOQ and 50×LOQ) yielded satisfactory recoveries for all tested analytes in the range of 83.01 to 110.18% with relative standard deviation (RSD)≤8.91. The method was successfully applied to field-incurred plum samples and dinotefuran and all metabolites were positively detected and quantified. In conclusion, we suggest that the method can be expanded to polar compounds having solvent and partitioning problems in any of the versions of QuEChERS.

Development and validation of modified QuEChERS method coupled with LC-MS/MS for simultaneous determination of cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite in various types of honey and royal jelly.

Over the past few decades, honey products have been polluted by different contaminants, such as pesticides, which are widely applied in agriculture. In this work, a modified EN - quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method was developed for the simultaneous quantification of pesticide residues, including cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite 2,4-dimethylaniline (2,4-DMA), in four types of honey (acacia, wild, chestnut, and manuka) and royal jelly. Samples were buffered with 0.2M dibasic sodium phosphate (pH 9), and subsequently, acetonitrile was employed as the extraction solvent. A combination of primary secondary amine (PSA) and C18 sorbents was used for purification prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) analysis. The estimated linearity measured at six concentration levels presented good correlation coefficients (R2)≥0.99. The recovery, calculated from three different spiking levels, was 62.06-108.79% in honey and 67.58-106.34% in royal jelly, with an RSD<12% for all the tested compounds. The matrix effect was also evaluated, and most of the analytes presented signal enhancement. The limits of quantification (LOQ) ranged between 0.001 and 0.005mg/kg in various samples. These are considerably lower than the maximum residue limits (MRL) set by various regulatory authorities. A total of 43 market (domestic and imported) samples were assayed for method application. Among the tested samples, three samples were tested positive (i.e. detected and quantified) only for cymiazole residues. The residues in the rest of the samples were detected but not quantified. We concluded that the protocol developed in this work is simple and versatile for the routine quantification of cymiazole, 2,4-DMA, fipronil, coumaphos, amitraz, and fluvalinate in various types of honey and royal jelly.

Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

Dual-specificity phosphatases (DUSPs) constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central ß-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

Decline patterns and risk assessment of 10 multi-class pesticides in young sprout amaranth (Amaranthus mangostanus) under greenhouse growing conditions.

The present study was designed to investigate the residual decline pattern and the risk assessment of 10 different class pesticides, namely azoxystrobin, boscalid, diazinon, diethofencarb, difenoconazole, etofenprox, flubendiamide, paclobutrazol, and pyraclostrobin in young vegetative amaranth (Amaranthus mangostanus) sprayed once or twice under greenhouse growing conditions. Field-incurred samples, collected at 3, 7, or 10 days after application of both treatments, were extracted and purified with the quick, easy, cheap, effective, rugged, and safe "QuEChERS" citrate-buffered method and analyzed with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) in positive ion mode. The linearity was satisfactory with determination coefficients (R 2) falling between 0.9817 and 0.9999 and limits of detection (LOD) and quantification (LOQ) values of 0.0007 and 0.002 mg/kg, respectively. The mean recovery rate at four spiking levels (equivalent to 5, 10, 50, and 100 × LOQ) ranged from 78.1 to 131.6% with a relative standard deviation (RSD) of < 11%. Substantial differences in the initial deposit between the tested analytes were observed and clearly indicated that the structure, as well as the initial concentration of applied products, greatly affected the residue deposit. From the obtained residual data, the provisional marginal maximum residue limits (MRLs) and the pre-harvest intervals (PHI) were proposed. Risk assessment was evaluated by comparing the theoretical maximum daily intake (TMDI) with the acceptable daily intake (ADI). Herein, the TMDI was lower than the ADI (TMDI/ADI ratio ≤ 80% set by the Korean Ministry of Food and Drug Safety) except for difenoconazole (80.92%, marginally higher), indicating that the vegetative amaranth is not hazardous and can be consumed safely by Korean consumers.

Crystal structures of two forms of the Acanthamoeba polyphaga mimivirus Rab GTPase.

Acanthamoeba polyphaga mimivirus (APMV) is a member of the family of giant viruses, harboring a 1,200 kbp genome within its 700 nm-diameter viral particle. The R214 gene of the APMV genome was recently shown to encode a homologue of the Rab GTPases, molecular switch proteins known to play a pivotal role in the regulation of membrane trafficking that were considered to exist only in eukaryotes. Herein, we report the first crystal structures of GDP- and GTP-bound forms of APMV Rab GTPase, both of which were determined at high resolution. An in-depth structural comparison of APMV Rab with each other and with mammalian Rab homologues led to an atomic-level elucidation of the inactive-active conformational change upon GDP/GTP exchange. APMV Rab GTPase exhibited considerable structural similarity to human Rab5, as previously predicted based on its amino acid sequence. However, it also contains unique structural features differentiating it from mammalian homologues, such as the functional substitution of a phenylalanine residue for the stabilization of the nucleotide's guanine base.

Sustainability of CD24 expression, cell proliferation and migration, cisplatin-resistance, and caspase-3 expression during mesenchymal-epithelial transition induced by the removal of TGF-ß1 in A549 lung cancer cells.

Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor ß1 (TGF-ß1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-ß1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-ß1 (TGF-ß1-treated group) and MET was induced sequentially from the TGF-ß1-treated group by removing the TGF-ß1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-ß1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24low cells was reduced in the TGF-ß1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-ß1-treated group. These features were unaltered in the MET/return group when compared to the TGF-ß1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-ß1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-ß1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-ß1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-ß1.