Shifts in receptors during submergence of an encephalitic arbovirus.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

VLDLR and ApoER2 are receptors for multiple alphaviruses.

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

STAT3-iNOS Signaling Mediates EGFRvIII-Induced Glial Proliferation and Transformation.

Malignant gliomas, including glioblastoma multiforme, constitute the most common and aggressive primary brain tumors in adults. The transcription factor signal transducer and activator of transcription 3 (STAT3) plays an essential role in glioblastoma pathogenesis downstream of the major oncogenic protein epidermal growth factor receptor variant III (EGFRvIII). However, the critical gene targets of STAT3 that mediate EGFRvIII-induced glial transformation have remained unknown. Here, we identify inducible nitric oxide synthase (iNOS) as a novel target gene of STAT3 in EGFRvIII-expressing mouse astrocytes. Endogenous STAT3 occupies the endogenous iNOS promoter and stimulates iNOS transcription in EGFRvIII-expressing astrocytes. STAT3 does not appear to control iNOS transcription in astrocytes deficient in the major glioblastoma tumor suppressor protein phosphatase and tensin homolog (PTEN), suggesting that STAT3 regulates iNOS transcription specifically in EGFRvIII-expressing astrocytes. Importantly, inhibition of iNOS by distinct approaches, including knockdown by RNA interference, reduces cell population growth and invasiveness of EGFRvIII-expressing astrocytes. In addition, upon iNOS knockdown or administration of a small-molecule inhibitor of iNOS, EGFRvIII-expressing astrocytes form smaller tumors in vivo. These findings suggest that inhibition of iNOS may have potential therapeutic value for EGFRvIII-activated brain tumors.

Utility of Histologic and Histochemical Screening for 16S Ribosomal RNA Gene Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue for Bacterial Endocarditis.

OBJECTIVES: 16S ribosomal RNA (rRNA) sequencing is a powerful but expensive tool for the identification of bacteria in culture-negative endocarditis. Histologic criteria to screen formalin-fixed, paraffin-embedded (FFPE) specimens for testing are evaluated. METHODS: Sixty-eight cases of infective endocarditis and controls were histologically reviewed and analyzed by 16S rRNA gene sequencing. RESULTS: Sequencing identified a specific pathogenic organism in 33 (49%) of 68 cases with acute inflammation and in 0 of 10 controls (P = .004). Visualization of organisms by Gram or Grocott methenamine silver stains had the strongest association with positive sequencing, while antibiotic treatment effect and acid decalcification decreased sensitivity. Molecular identifications were concordant with blood culture results in 90% of the cases, and a positive sequencing result was obtained in approximately half of the cases with negative valve cultures. CONCLUSIONS: Histologic screening criteria are extremely helpful for identifying cases likely to be positive by molecular testing and can provide significant cost savings in filtering out low-yield specimens.

The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis.

While 18 putative RNA helicases are involved in ribosome biogenesis in Saccharomyces cerevisiae, their enzymatic properties have remained largely biochemically uncharacterized. To better understand their function, we examined the enzymatic properties of Dpb8, a DExD/H box protein previously shown to be required for the synthesis of the 18S rRNA. As expected for an RNA helicase, we demonstrate that recombinant Dbp8 has ATPase activity in vitro, and that this activity is dependent on an intact ATPase domain. Strikingly, we identify Esf2, a nucleolar putative RNA binding protein, as a binding partner for Dbp8, and show that it enhances Dbp8 ATPase activity by decreasing the K(M) for ATP. Thus, we have uncovered Esf2 as the first example of a protein co-factor that has a stimulatory effect on a nucleolar RNA helicase. We show that Esf2 can bind to pre-rRNAs and speculate that it may function to bring Dbp8 to the pre-rRNA, thereby both regulating its enzymatic activity and guiding Dbp8 to its site of action.

CAR-T Cell Therapies From the Transfusion Medicine Perspective.

The use of chimeric antigen receptor (CAR)-T cell therapy for the treatment of hematologic malignancies has generated significant excitement over the last several years. From a transfusion medicine perspective, the implementation of CAR-T therapy as a potential mainstay treatment for not only hematologic but also solid-organ malignancies represents a significant opportunity for growth and expansion. In this review, we will describe the rationale for the development of genetically redirected T cells as a cancer therapeutic, the different elements that are required to engineer these cells, as well as an overview of the process by which patient cells are harvested and processed to create and subsequently validate CAR-T cells. Finally, we will briefly describe some of the toxicities and clinical efficacy of CAR-T cells in the setting of patients with advanced malignancy.