Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this ...lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic KrasG12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia PanIN or Intraductal papillary mucinous neoplasm IPMN with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.
Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif ...and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.
•Studied 3-residue serine/threonine motif in N-termini of AAV VP1 and VP2.•Motif may impact virus formation, cell binding and entry, viral transcription.•One AAV9 S/T variant has altered behavior in C57BL/6 mice.
In January 2019, instant noodle giant Nissin Foods released two animated advertisements online featuring Naomi Osaka, which elicited backlash over “whitewashing” the multiracial professional tennis ...player who represents Japan. Although Nissin has since pulled the advertisements and officially apologized for portraying Osaka as lighter-skinned, underlying these issues are also those of gender, especially its intersections with race/ethnicity, nation, and sport in Japanese media. Employing discourse analysis of these two advertisements, this article examines how Nissin represented Osaka and what ideologies these representations reflect in Japanese society. Drawing on intersectional and transnational feminist cultural studies approaches, we argue that the advertisements’ representation of Osaka and the ensuing controversy reflect racialized and gendered ideologies in an allegedly homogeneous Japanese society, informed by local media and popular cultures that regularly portray “racially neutral” characters, celebrate lighter-skinned hāfu (half; multiracial) women, and diminish sportswomen’s athletic abilities. This article contributes to communication and sport studies by situating Osaka within broader contexts of how hāfu and sporting women are depicted in Japanese media and how elite sportswomen and multiracial athletes are portrayed in international media. Our article concludes by offering two suggestions on how to research multiracial sportswomen in the media.
Mutations in parkin are currently recognized as the most common cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for the ...development of the more common, sporadic form of Parkinson's disease (PD). Supporting this, we have recently demonstrated that parkin solubility in the human brain becomes altered with age. As parkin apparently functions as a broad-spectrum neuroprotectant, the resulting decrease in the availability of soluble parkin with age may underlie the progressive susceptibility of the brain to stress. Interestingly, we also observed that many familial-PD mutations of parkin alter its solubility in a manner that is highly reminiscent of our observations with the aged brain. The converging effects on parkin brought about by aging and PD-causing mutations are probably not trivial and suggest that environmental modulators affecting parkin solubility would increase an individual's risk of developing PD. Using both cell culture and in vivo models, we demonstrate here that several PD-linked stressors, including neurotoxins (MPP+, rotenone, 6-hydroxydopamine), paraquat, NO, dopamine and iron, induce alterations in parkin solubility and result in its intracellular aggregation. Furthermore, the depletion of soluble, functional forms of parkin is associated with reduced proteasomal activities and increased cell death. Our results suggest that exogenously introduced stress as well as endogenous dopamine could affect the native structure of parkin, promote its misfolding, and concomitantly compromise its protective functions. Mechanistically, our results provide a link between the influence of environmental and intrinsic factors and genetic susceptibilities in PD pathogenesis.
The biological impact of exogenous, alternating electric fields (AEFs) and direct-current electric fields has a long history of study, ranging from effects on embryonic development to influences on ...wound healing. In this article, we focus on the application of electric fields for the treatment of cancers. In particular, we outline the clinical impact of tumor treating fields (TTFields), a form of AEFs, on the treatment of cancers such as glioblastoma and mesothelioma. We provide an overview of the standard mechanism of action of TTFields, namely, the capability for AEFs (e.g., TTFields) to disrupt the formation and segregation of the mitotic spindle in actively dividing cells. Though this standard mechanism explains a large part of TTFields’ action, it is by no means complete. The standard theory does not account for exogenously applied AEFs’ influence directly upon DNA nor upon their capacity to alter the functionality and permeability of cancer cell membranes. This review summarizes the current literature to provide a more comprehensive understanding of AEFs’ actions on cell membranes. It gives an overview of three mechanistic models that may explain the more recent observations into AEFs’ effects: the voltage-gated ion channel, bioelectrorheological, and electroporation models. Inconsistencies were noted in both effective frequency range and field strength between TTFields versus all three proposed models. We addressed these discrepancies through theoretical investigations into the inhomogeneities of electric fields on cellular membranes as a function of disease state, external microenvironment, and tissue or cellular organization. Lastly, future experimental strategies to validate these findings are outlined. Clinical benefits are inevitably forthcoming.
This article explores the mediation of androgynous bodies and styles in contemporary Japan by mapping the relationship between dansō (female-to-male crossdressing) and genderless (jendāresu). Dansō ...refers to gender-crossing practices usually by individuals who are assigned female at birth, whereas genderless is a mode of fashion emerging in 2010 which theoretically denotes styles that do not distinguish between genders. Drawing on media coverage of dansō individuals and genderless joshi (girls) in the 2010s, supplemented by ethnographic research conducted in a Tokyo dansō cafe-and-bar, I argue that these individuals embraced genderbending practices before being named as "doing" dansō or genderless. Through their distinct but related androgynous practices, they construct alternative identities and ways of being and retrospectively become interpellated as "dansō" and "genderless." Further, I suggest that dansō and genderless not only allow us to rethink the gender binary, particularly in queer studies and transgender studies in a transnational context, but also the connections between style and gender and sexual subjectivities. Placing dansō and genderless on a spectrum instead of considering the two as separate phenomena opens up new conversations about androgynous bodies at the intersections of queer studies, fashion studies, and cultural studies in Asia.
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BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these ...subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to “resensitize” upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.
► TARP and cornichon subunits synergistically modulate hippocampal AMPA receptors ► AMPA receptor resensitization induced by TARP subunits
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would ...allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By codelivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34+ hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγnull mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34+ cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.
•Delivery of ZFNs and donor templates results in high levels of gene correction in human CD34+ cells from multiple sources, including SCD BM.•Modified CD34+ cells are capable of engrafting immunocompromised NSG mice and produce cells from multiple lineages.
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with ...heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, ...its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.
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Guenther et al. have developed a protease-activatable AAV provector to target diseased tissue microenvironments exhibiting elevated extracellular protease activity. The provector delivers transgenes to high-MMP-activity regions of the damaged heart following a myocardial infarction, with concomitant decreased delivery to many off-target organs, including the liver.
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