Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains.

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

Effects of particle size on toxicity, bioaccumulation, and translocation of zinc oxide nanoparticles to bok choy (Brassica chinensis L.) in garden soil.

The indiscriminate use of zinc oxide nanoparticles (ZnO NPs) in daily life can lead to their release into soil environment. These ZnO NPs can be taken up by crops and translocated to their edible part, potentially causing risks to the ecosystem and human health. In this study, we conducted pot experiments to determine phytotoxicity, bioaccumulation and translocation depending on the size (10 - 30 nm, 80 - 200 nm and 300 nm diameter) and concentration (0, 100, 500 and 1000 mg Zn/kg) of ZnO NPs and Zn ion (Zn2+) in bok choy, a leafy green vegetable crop. After 14 days of exposure, our results showed that large-sized ZnO NPs (i.e., 300 nm) at the highest concentration exhibited greater phytotoxicity, including obstruction of leaf and root weight (42.5 % and 33.8 %, respectively) and reduction of chlorophyll a and b content (50.2 % and 85.2 %, respectively), as well as changes in the activities of oxidative stress responses compared to those of small-sized ZnO NPs, although their translocation ability was relatively lower than that of smaller ones. The translocation factor (TF) values decreased as the size of ZnO NPs increased, with TF values of 0.68 for 10 - 30 nm, 0.55 for 80 - 200 nm, and 0.27 for 300 nm ZnO NPs, all at the highest exposure concentration. Both the results of micro X-ray fluorescence (µ-XRF) spectrometer and bio-transmission electron microscopy (bio-TEM) showed that the Zn elements were mainly localized at the edges of leaves exposed to small-sized ZnO NPs. However, the Zn elements upon exposure to large-sized ZnO NP were primarily observed in the primary veins of leaves in the µ-XRF data, indicating a limitation in their ability to translocate from roots to leaves. This study not only advances our comprehension of the environmental impact of nanotechnology but also holds considerable implications for the future of sustainable agriculture and food safety.

Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis.

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

BCL-2 family protein BOK is a positive regulator of uridine metabolism in mammals.

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

Mass spectrometry and synchrotron-FTIR microspectroscopy reveal the anti-inflammatory activity of Bua Bok extracts.

INTRODUCTION: Bua Bok or Centella asiatica (CA) is an Asian vegetable with anti-inflammatory benefits. Asiaticoside, asiatic acid, madecassoside and madecassic have been characterised as major active ingredients with a wide range of pharmacological advantages. In manufacturing processes, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) are used to routinely determine the active compounds in raw materials. OBJECTIVES: This research aims to explore anti-inflammatory properties, characterise metabolites and observe the biochemical changes of the inflammatory induced macrophages after pretreatment with the potential extracted fractions. METHODS: Bua Bok leaf extracts were prepared. Macrophages were pretreated with non-toxic fractions to determine the anti-inflammatory action. Tentative metabolites of effective fractions were identified by LC-MS. Synchrotron fourier-transform infrared (S-FTIR) microspectroscopy was utilised to observe the biochemical change of the lipopolysaccharide (LPS)-induced cells after pretreatment with potential fractions. RESULTS: Fractions of ethyl acetate, 30% and 100% ethanol highly increased the nitrile scavenging and suppressed the function of phospholipase A2 . Fractions of 70% and 100% ethanol strongly decreased nitric oxide production. The comparison of 39 chemical compounds was presented. The change of proteins was improved after pretreatment of macrophages with fraction 70% ethanol. Fraction of 100% ethanol revealed the lipid accumulation was lower than 70% ethanol and diclofenac. CONCLUSION: While the anti-inflammatory actions of 70% and 100% ethanol were similar. S-FTIR expressed they inhibited inflammatory response with the distinct features of biomolecules. The S-FTIR, LC-MS and biological assay confidently provided the efficient strategies to inform the advantage of herbal extract on cellular organisation instead of a single compound.

Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP3Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP3R1, focusing on the ∼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914-1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP3R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP3R1-derived peptides revealed high affinity binding to amino acids 1898-1940 of IP3R1 (Kd ∼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP3R1 interaction was demonstrated by the ability of IP3R1 or Bok to recruit transmembrane domain-free Bok or IP3R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP3R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.

Development of a nanobody-based ELISA for the detection of the insecticides cyantraniliprole and chlorantraniliprole in soil and the vegetable bok choy.

Cyantraniliprole and chlorantraniliprole are anthranilic diamide insecticides acting on ryanodine receptors. In this study, two camel-derived nanobodies (Nbs, named C1 and C2) recognizing cyantraniliprole as well as chlorantraniliprole were generated. C1-based enzyme-linked immunosorbent assays (ELISAs) for the detection of the two insecticides were developed. The half-maximum signal inhibition concentrations (IC50) of cyantraniliprole and chlorantraniliprole by ELISA were 1.2 and 1.5 ng mL-1, respectively. This assay was employed to detect these two insecticides in soil and vegetables. The average recoveries of cyantraniliprole from both bok choy (Brassica chinensis L.) and soil samples were 90-129%, while those of chlorantraniliprole were in a range of 89-120%. The insecticide residues in soil and bok choy, which were collected from plots sprayed with cyantraniliprole and chlorantraniliprole, were simultaneously detected by the resulting ELISA and a high-performance liquid chromatography (HPLC) method, showing a satisfactory correlation. Higher concentrations of chlorantraniliprole than cyantraniliprole were detected in soil and vegetables, which indicates the longer persistence of chlorantraniliprole in the environment.

Loss of BOK Has a Minor Impact on Acetaminophen Overdose-Induced Liver Damage in Mice.

Acetaminophen (APAP) is one of the most commonly used analgesic and anti-pyretic drugs, and APAP intoxication is one of the main reasons for liver transplantation following liver failure in the Western world. While APAP poisoning ultimately leads to liver necrosis, various programmed cell death modalities have been implicated, including ER stress-triggered apoptosis. The BCL-2 family member BOK (BCL-2-related ovarian killer) has been described to modulate the unfolded protein response and to promote chemical-induced liver injury. We therefore investigated the impact of the loss of BOK following APAP overdosing in mice. Surprisingly, we observed sex-dependent differences in the activation of the unfolded protein response (UPR) in both wildtype (WT) and Bok-/- mice, with increased activation of JNK in females compared with males. Loss of BOK led to a decrease in JNK activation and a reduced percentage of centrilobular necrosis in both sexes after APAP treatment; however, this protection was more pronounced in Bok-/- females. Nevertheless, serum ALT and AST levels of Bok-/- and WT mice were comparable, indicating that there was no major difference in the overall outcome of liver injury. We conclude that after APAP overdosing, loss of BOK affects initiating signaling steps linked to ER stress, but has a more minor impact on the outcome of liver necrosis. Furthermore, we observed sex-dependent differences that might be worthwhile to investigate.

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.

Placental cell death patterns exhibit differences throughout gestation in two strains of laboratory mice.

Cell death is an essential physiological process required for the proper development and function of the human placenta. Although the mouse is a commonly used animal model for development studies, little is known about the extent and distribution of cell death in the mouse placenta throughout development and its physiological relevance. In the present study, we report the results of a systematic and quantitative assessment of cell death patterns in the placentae of two strains of laboratory mice commonly used for developmental studies-ICR and C57Bl/6. TUNEL staining revealed that ICR and C57Bl/6 placentae exhibited similar cell death patterns to those reported in human placentae during pregnancy, with comparatively infrequent death observed during early gestation, which increased and became more organized towards term. Interestingly, when comparing strain differences, increased cell death was observed in almost all regions of the inbred C57Bl/6 placentae compared to the outbred ICR strain. Finally, since Bcl-2 ovarian killer (Bok) has been reported to be a key player in human placental cell death, we examined its expression in murine placentae throughout gestation. Bok protein expression was observed in all placental regions and increased towards term in both strains. The results of this study indicate that although strain-specific differences in placental cell death exist, the overall rates and patterns of cell death during murine placentation parallel those previously described in humans. Thus, the murine placenta is a useful model to investigate molecular pathways involved in cell death signaling during human placentation.

BOK promotes erythropoiesis in a mouse model of myelodysplastic syndrome.

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by cytopenia and intramedullary apoptosis. BCL-2 Ovarian Killer (BOK) is a pro-apoptotic member of the BCL-2 family of proteins which, when stabilized from endoplasmic reticulum-associated degradation (ERAD), induces apoptosis in response to ER stress. Although ER stress appropriately activates the unfolded protein response (UPR) in BOK-disrupted cells, the downstream effector signaling that includes ATF4 is defective. We used Nup98-HoxD13 (NHD13) transgenic mice to evaluate the consequences of BOK loss on hematopoiesis and leukemogenesis. Acute myeloid leukemia developed in 36.7% of NHD13 mice with a Bok gene knockout between the age of 8 and 13 months and presented a similar overall survival to the NHD13 mice. The loss of BOK exacerbated anemia in NHD13 mice, and NHD13/BOK-deficient mice exhibited significantly lower hemoglobin, lower mean cell hemoglobin concentration, and higher mean cell volume than NHD13 mice. Hematopoietic progenitor cell assays revealed a decreased amount of erythroid progenitor stem cells (BFU-E) in the bone marrow of NHD13-transgenic/BOK-deficient mice. RT-qPCR analysis demonstrated decreased mean value of ATF4 in the erythroid progenitors of NHD13 and NHD13/BOK-deficient mice. Our results suggest that in addition to induction of apoptosis in response to ER stress, BOK may regulate erythropoiesis when certain erythroid progenitors experience cell stress.

Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak.

The pro-apoptotic multidomain Bcl-2 proteins Bax and Bak (also known as BAK1) are considered the gatekeepers of the intrinsic pathway of apoptosis by triggering the mitochondrial release of cytochrome c The role of the third Bax- and Bak-homologous multidomain protein Bok, however, is still unresolved. As cells doubly deficient for Bax and Bak are largely resistant to various apoptotic stimuli, it has been proposed that Bok is either dispensable for apoptosis or that its role is dependent on Bax and Bak. Here, we demonstrate, in several cell systems, that Bok efficiently induces cytochrome c release and apoptosis even in the complete absence of both Bak and Bax. Moreover, modulation of endogenous Bok levels affects the apoptosis response. By RNA interference and targeted deletion of the Bok gene, we demonstrate that Bok can significantly influence the apoptotic response to chemotherapeutic drugs in ovarian carcinoma cells. Hence, our results not only establish Bok as a Bak- and Bax-independent apoptosis inducer, but also suggest a potential impact of Bok expression in ovarian cancer therapy.

BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress.

B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok(-/-) and even Bax(-/-)Bok(-/-) and Bak(-/-)Bok(-/-) mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax(-/-)Bak(-/-) mice despite its widespread expression. Here, we independently developed Bok(-/-) mice and found that Bok(-/-) cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok(-/-) mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok(-/-) mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok(-/-) cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok(-/-) cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing--to our knowledge--the first bona fide apoptotic defect linked to Bok deletion.

BOK displays cell death-independent tumor suppressor activity in non-small-cell lung carcinoma.

As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFß-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

Whole-exome sequencing of duodenal adenocarcinoma identifies recurrent Wnt/ß-catenin signaling pathway mutations.

BACKGROUND: Genomic alterations of small bowel cancers remain poorly understood due to the rarity of these diseases. In the current study, the authors report the identification of somatic mutations from patients with duodenal adenocarcinoma by whole-exome sequencing. METHODS: Whole-exome sequencing and follow-up analysis were conducted in 12 matched tumor-normal tissue duodenal adenocarcinoma tissue pairs to examine the genetic characteristics of this disease. Somatic mutations (single-nucleotide variants and short insertion/deletions) were obtained and filtered and then searched for recurrently mutated genes and pathways. RESULTS: An excess of C-to-T transitions at the CpG dinucleotide was observed in the substitution of bases. The authors identified recurrent mutations in tumor protein p53 (TP53), KRAS, catenin (cadherin-associated protein) ß-1 (CTNNB1), AT-rich interactive domain 2 (ARID2), adenomatous polyposis coli (APC), erb-b2 receptor tyrosine kinase 2 (ERBB2), ARID1A, cadherin-related family member 1 (CDHR1), NRAS, Bcl-2-related ovarian killer (BOK), radial spoke head 14 homolog (chlamydomonas) (RTDR1), cell division cycle 27 (CDC27), catalytic subunit of phosphoinositide-3-kinase (PIK3CA), and SMAD family member 4 (SMAD4). Pathway scan indicated that the Wnt signaling pathway, regulation of the actin cytoskeleton pathway, ErbB signaling pathway, and the pathway of focal adhesion were the most extensively affected pathways. CONCLUSIONS: This genomic characterization of duodenal adenocarcinoma provides researchers with insight into its somatic landscape and highlights the vital role of the Wnt/ß-catenin signaling pathway. The study data also indicate that duodenal adenocarcinomas have a genetic resemblance to gastric and colorectal cancers. These discoveries may benefit the future development of molecular diagnosis and personalized therapies. Cancer 2016;122:1689-96. © 2016 American Cancer Society.

Identification of the Bcl-2 family protein gene BOK from orange-spotted grouper (Epinephelus coioides) involved in SGIV infection.

Apoptosis plays vital roles in many physiological process and immune response. BOK is one of the central regulators in apoptosis. In this study, a new BOK homolog (Ec-BOK) was cloned and characterized from Orange-spotted grouper, Epinephelus coioides. Ec-BOK encoded a 210 amino acid peptides which shared 97% identity to Stegastes partitus BOK protein, contained four BH domains and one transmembrane region. Ec-BOK widely expressed in all analyzed tissues with the higher expressions in kidney and spleen. Its expression level was up-regulated after SGIV infection in vitro. Further analysis revealed that overexpression of Ec-BOK inhibited viral genes transcriptions and virus replication in fish cell. Our findings suggested that Ec-BOK might play a role in the immune response against virus.

Molecular profiles and pathogen-induced transcriptional responses of prawn B cell lymphoma-2 related ovarian killer protein (BOK).

In this study, we have reported a molecular characterization of the first B cell lymphoma-2 (BCL-2) related ovarian killer protein (BOK) from freshwater prawn Macrobrachium rosenbergii (Mr). BOK is a novel pro-apoptotic protein of the BCL-2 family that entails in mediating apoptosis to remove cancer cells. A cDNA sequence of MrBOK was identified from the prawn cDNA library and its full length was obtained by internal sequencing. The coding region of MrBOK yields a polypeptide of 291 amino acids. The analysis revealed that MrBOK contains a transmembrane helix at V(261)-L(283) and a putative BCL-2 family domain at V(144)-W(245). MrBOK also possessed four putative BCL-2 homology domains including BH1, BH2, BH3 and weak BH4. The BH3 contains 21 binding sites and among them five residues are highly conserved with the aligned BOK proteins. The homology analysis showed that MrBOK shared maximum similarity with the Caligus rogercresseyi BOK A. The topology of the phylogenetic tree was classified into nine sister groups which includes BOK, BAK, BAX, BAD, BCL-2, BCL-XL, NR13 and MCL members. The BOK protein group further sub-grouped into vertebrate and invertebrate BOK, wherein MrBOK located within insect monophyletic clad of invertebrate BOK. The secondary structural analysis showed that MrBOK contains 11 α-helices (52.2%) which are connected over random coils (47.7%). The 3D structure of MrBOK showed three central helices (α6, α7 and α8) which formed the core of the protein and are flanked on one side by α1, α2 and α3, and on the other side by α4, α5 and α11. MrBOK mRNA is expressed most abundantly (P < 0.05) in ovary compared to other tissues taken for analysis. Hence ovary was selected to study the possible roles of MrBOK mRNA regulation upon bacterial (Aeromonas hydrophila and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodovirus] infection. During bacterial and viral infection, the highest MrBOK mRNA transcription was varied at different time points. In bacterial infected ovary tissue, the highest mRNA expression was at 24 h post-infection, whereas in viral infection, the expression was highest at 48 h post-infection. Thus we can conclude that MrBOK functions as an apoptotic protein in intracellular programmed cell-death pathway to counteract the anti-apoptotic proteins released by bacterial and viral pathogens at the time of infection. This is the first study that emphasizes the importance of BOK during bacterial and viral infection in crustacean.

The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage.

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895-1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.