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We report a simple, cost-effective, scalable and efficient method for creating transgenic Caenorhabditis elegans that requires minimal hands-on time. The method combines biolistic bombardment with selection for transgenics that bear a hygromycin B resistance gene on agar plates supplemented with hygromycin B, taking advantage of our observation that hygromycin B is sufficient to kill wild-type C. elegans at very low concentrations. Crucially, the method provides substantial improvements in the success of bombardments for isolating transmitting strains, the isolation of multiple independent strains, and the isolation of integrated strains: 100% of bombardments in a large data set yielded transgenics; 10 or more independent strains were isolated from 84% of bombardments, and up to 28 independent strains were isolated from a single bombardment; 82% of bombardments yielded stably transmitting integrated lines with most yielding multiple integrated lines. We anticipate that the selection will be widely adopted for C. elegans transgenesis via bombardment, and that hygromycin B resistance will be adopted as a marker in other approaches for manipulating, introducing or deleting DNA in C. elegans.
Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.
The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta. Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1, which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.
Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.
Naringenin (Nar), a common dietary flavonoid abundantly present in fruits, vegetables, and Chinese herbs, is believed to possess strong anti-inflammatory properties and to modulate hepatic apolipoprotein and lipid synthesis. However, there are no reports describing Nar's effects on the hepatitis B virus protein X (HBx) -induced hepatic steatosis, and the detailed molecular mechanisms of the compound's effects are still unclear.
Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.
Orally administered fungal vaccines show promise for the prevention of infectious diseases. Edible mushrooms are deemed appropriate hosts to produce oral vaccines due to their low production cost and low risk of gene contamination. However, their low expression level of antigens has limited the potential development of oral vaccines using mushrooms. The low expression level might result from impurity of the transgenic mycelia since dikaryotic mycelia are commonly used as transformation materials. In this study, stable transgenic hepatitis B virus surface antigen (HBsAg) in Flammulina velutipes transformants was obtained by Agrobacterium-mediated transformation, followed by fruiting and basidiospore mating. The formation of HBsAg was detected by western blot analysis. The expression levels of HBsAg in transgenic F. velutipes fruiting bodies were (129.3±15.1), (110.9±1.7) and (161.1±8.5) ng/g total soluble protein. However, the values may be underestimated due to incomplete protein extraction. Two of the four pigs in the experimental group produced positive anti-HBsAg-specific IgG after being fed the HBsAg transgenic F. velutipes fruiting bodies for 20 weeks, while no anti-HBsAg antibody was detected in the control group. One of the positive pigs had HBsAg titres of 5.36 and 14.9 mIU/mL in weeks 10 and 14, respectively, but expression faded thereafter. The other positive pig displayed HBsAg titres of 9.75, 17.86 and 39.87 mIU/mL in weeks 14, 18 and 20, respectively. The successful immunogenicity in pigs fed transgenic F. velutipes fruiting bodies demonstrated the potential of using the fungus as an oral vaccine.
Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.
Drug resistance is a major challenge for all oncological treatments that involve the use of cytotoxic agents. Recent therapeutic alternatives cannot circumvent the ability of cancer cells to adapt or alter the natural selection of resistant cells, so the problem persists. In neuroblastoma, recurrence can occur in up to 50% of high-risk patients. Therefore, the identification of novel therapeutic targets capable of modulating survival or death following classical antitumor interventions is crucial to address this problem. In this study, we investigated the role of the P2X7 receptor in chemoresistance. Here, we elucidated the contributions of P2X7 receptor A and B isoforms to neuroblastoma chemoresistance, demonstrating that the B isoform favors resistance through a combination of mechanisms involving drug efflux via MRP-type transporters, resistance to retinoids, retaining cells in a stem-like phenotype, suppression of autophagy, and EMT induction, while the A isoform has opposite and complementary roles.
Many individuals are infected with hepatitis B virus (HBV) worldwide, and this virus is commonly controlled by treatments with interferon (IFN)-alpha and nucleoside analogues (NA). However, the complete elimination of HBV by these treatments is difficult and, thus, the development of new treatments is needed. Host immune responses are closely involved in the elimination of HBV, suggesting the usefulness of immunotherapy. In the present study, we attempted to identify novel cytotoxic T-lymphocyte (CTL) epitopes that are useful for immunotherapy against HBV.
Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.
Aurora B kinase plays an essential role in chromosome bi-orientation, which is a prerequisite for equal segregation of chromosomes during mitosis. However, it remains largely unclear whether centromere-localized Aurora B is required for faithful chromosome segregation. Here we show that histone H3 Thr-3 phosphorylation (H3pT3) and H2A Thr-120 phosphorylation (H2ApT120) can independently recruit Aurora B. Disrupting H3pT3-mediated localization of Aurora B at the inner centromere impedes the decline in H2ApT120 during metaphase and causes H2ApT120-dependent accumulation of Aurora B at the kinetochore-proximal centromere. Consequently, silencing of the spindle assembly checkpoint (SAC) is delayed, whereas the fidelity of chromosome segregation is negligibly affected. Further eliminating an H2ApT120-dependent pool of Aurora B restores proper timing for SAC silencing but increases chromosome missegregation. Our data indicate that H2ApT120-mediated localization of Aurora B compensates for the loss of an H3pT3-dependent pool of Aurora B to correct improper kinetochore-microtubule attachments. This study provides important insights into how centromeric Aurora B regulates SAC and kinetochore attachment to microtubules to ensure error-free chromosome segregation.
Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.
Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.
Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.
Membrane lipid biosynthesis is a complex process that takes place in various intracellular compartments. Glycosylphosphatidylinositol (GPI), a lipid involved in membrane anchoring of some proteins, is synthesized by the PIG enzymes. Most PIGs are localized to the endoplasmic reticulum (ER), but Drosophila PIG-B (DmPIG-B) is localized to the nuclear envelope (NE). To determine whether the NE localization of DmPIG-B is functionally important, we defined the determinants of localization and generated an ER-localized form, denoted DmPIG-B[ER]. The enzymatic activity of DmPIG-B[ER] was comparable to that of NE-localized DmPIG-B[NE]. Expression of DmPIG-B[ER] inefficiently rescued the lethality of the PIG-B mutant, whereas DmPIG-B[NE] rescued this lethality fully. DmPIG-B[ER] was preferentially degraded by lysosomes, suggesting that the NE localization is essential for function and stability of the protein. In addition, we found that the region of the ER proximal to the NE is the site of translation of GPI-anchored proteins and addition of GPI. Thus, the NE and proximal ER may provide a platform for efficient GPI anchoring.
The BCL6 proto-oncogene encodes a transcriptional repressor required for the development of germinal centers (GCs) and implicated in the pathogenesis of GC-derived B cell lymphoma. Understanding the precise role of BCL6 in normal GC formation and in lymphomagenesis depends on the identification of genes that are direct targets of its transcriptional repression. Here we report that BCL6 directly controls the expression of B7-1/CD80, a costimulatory receptor involved in B-T cell interactions critical for the development of T cell-mediated antibody responses. Upon CD40 signaling, transcription of the CD80 gene is induced by the nuclear factor (NF)-kappaB transcription factor. Our results show that BCL6 prevents CD40-induced expression of CD80 by binding its promoter region in vivo and suppressing its transcriptional activation by NF-kappaB. Consistent with a physiologic role for BCL6 in suppressing CD80, the expression of these two genes is mutually exclusive in B cells, and BCL6-defective mice show increased expression of CD80 in B cells. The results suggest that BCL6 may directly control the ability of B cell to interact with T cells during normal GC development. In addition, these findings imply that T-B cell interactions may be disrupted in B cell lymphoma expressing deregulated BCL6 genes.
B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.
IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFβ-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin-1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer.
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