On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization.

Biosafety, biosecurity, logistical, political, and technical considerations can delay or prevent the wide dissemination of source material containing viable virus from the geographic origin of an outbreak to laboratories involved in developing medical countermeasures (MCMs). However, once virus genome sequence information is available from clinical samples, reverse-genetics systems can be used to generate virus stocks de novo to initiate MCM development. In this study, we developed a reverse-genetics system for natural isolates of Ebola virus (EBOV) variants Makona, Tumba, and Ituri, which have been challenging to obtain. These systems were generated starting solely with in silico genome sequence information and have been used successfully to produce recombinant stocks of each of the viruses for use in MCM testing. The antiviral activity of MCMs targeting viral entry varied depending on the recombinant virus isolate used. Collectively, selecting and synthetically engineering emerging EBOV variants and demonstrating their efficacy against available MCMs will be crucial for answering pressing public health and biosecurity concerns during Ebola disease (EBOD) outbreaks.

Nanosized copper(ii) oxide/silica for catalytic generation of nitric oxide from S-nitrosothiols.

Nitric oxide NO, mediates inflammatory and thrombotic processes and designing biomaterials capable of releasing NO in contact with biological tissues is considered to be a major factor aimed at improving their bio- and haemocompatibility and antibacterial properties. Their NO-releasing capacity however is limited by the amount of the NO-containing substance incorporated in the bulk or immobilised on the surface of a biomaterial. An alternative approach is based on the design of a material generating nitric oxide from endogenous NO bearing metabolites by their catalytic decomposition. It offers, at least in theory, an unlimited source of NO for as long as the material remains in contact with blood and the catalyst maintains its activity. In this paper we studied the catalytic properties of novel nanostructured CuO/SiO2 catalysts in generating NO by decomposition of S-nitrosoglutathione (GSNO) in vitro. CuO/SiO2 catalysts with different CuO loadings were synthesized by chemisorption of copper(ii) acetylacetonate on fumed nanosilica followed by calcination. CuO content was controlled by a number of chemisorption-calcination cycles. Fourier-transform infrared spectroscopy and thermogravimetric analysis confirmed the formation of CuO/SiO2 nanoparticles (NPs) with particle size of CuO phase in the range from 71 to 88 nm. Scanning electron microscopy images revealed a uniform distribution of NPs without their sintering or agglomeration. All the materials of the CuO/SiO2 NP series exhibited NO-generating activity from GSNO confirmed by the Griess assay and by measuring the concentration of nitrite and nitrate anions in model solutions such as phosphate buffered saline and bovine serum. This activity is dependent on the material specific surface area and CuO exposure on the surface rather than CuO bulk content. The rate of NO production increased at higher initial concentration of the NO-bearing substrate studied in the range between 0.01 mM and 1.0 mM RSNO, which covers its physiological level. CuO/SiO2 NPs can be used to design polymers with NO generating properties at blood-biomaterial interface which are expected to have improved biocompatibility thus enhancing their potential for medical applications such as surgical tubing, peripheral venous catheters, auxiliary blood circulation devices and drug-eluting balloons.

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/ß/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

Critical role for cholesterol in Lassa fever virus entry identified by a novel small molecule inhibitor targeting the viral receptor LAMP1.

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.

Identification of Potent Ebola Virus Entry Inhibitors with Suitable Properties for in Vivo Studies.

Previous studies identified an adamantane dipeptide piperazine 3.47 that inhibits Ebola virus (EBOV) infection by targeting the essential receptor Niemann-Pick C1 (NPC1). The physicochemical properties of 3.47 limit its potential for testing in vivo. Optimization by improving potency, reducing hydrophobicity, and replacing labile moieties identified 3.47 derivatives with improved in vitro ADME properties that are also highly active against EBOV infection, including when tested in the presence of 50% normal human serum (NHS). In addition, 3A4 was identified as the major cytochrome P450 isoform that metabolizes these compounds, and accordingly, mouse microsome stability was significantly improved when tested in the presence of the CYP3A4 inhibitor ritonavir that is approved for clinical use as a booster of anti-HIV drugs. Oral administration of the EBOV inhibitors with ritonavir resulted in a pharmacokinetic profile that supports a b.i.d. dosing regimen for efficacy studies in mice.

Biochemical Basis for Increased Activity of Ebola Glycoprotein in the 2013-16 Epidemic.

Ebola virus (EBOV) infection is characterized by sporadic outbreaks caused by zoonotic transmission. Fixed changes in amino acid sequence, such as A82V in the EBOV glycoprotein (GP) that occurred early in the 2013-16 epidemic, are suspected to confer a selective advantage to the virus. We used biochemical assays of GP function to show that A82V, as well as a polymorphism in residue 544 identified in other outbreaks, enhances infection by decreasing the threshold for activation of membrane fusion activity triggered by the host factors cathepsin B and Niemann-Pick C1. Importantly, the increase in infectivity comes with the cost of decreased virus stability. Thus, emergence of a virus GP with altered properties that can affect transmission and virulence may have contributed to the severity and scope of the 2013-16 EBOV epidemic.

Rasburicase-Induced Methemoglobinemia in a Patient with Aggressive Non-Hodgkin's Lymphoma.

BACKGROUND: Rasburicase is a recombinant urate oxidase enzyme that converts uric acid to allantoin (an inactive and soluble metabolite that is readily excreted in urine). It is used for the management of tumor lysis syndrome (TLS) in cancer patients receiving chemotherapy. Although rasburicase is a generally safe and effective treatment, it can be associated with the rare and potentially severe complication of methemoglobinemia. Here, we report a case of rasburicase-induced methemoglobinemia in a patient who was diagnosed with aggressive non-Hodgkin's lymphoma. CASE REPORT: A 74-year-old man with aggressive non-Hodgkin's lymphoma was admitted for initiation of chemotherapy. Upon admission, the patient was found to have hyperkalemia, hyperuricemia, hyperphosphatemia, elevated LDH levels, and acute renal failure. As a result, he was diagnosed with TLS. Rasburicase 6 mg was administered intravenously over a period of 30 min to treat TLS. Later, methemoglobinemia developed, with requirements for oxygen supplementation. Multiple units of packed red blood cells were transfused for recurrent significant anemia secondary to his cancer co-morbidity. The patient was tested for glucose-6 phosphate dehydrogenase (G6PD) deficiency, which returned negative; therefore, methylene blue was considered. After transfusion, the methemoglobin level normalized over the course of a few days, and the oxygen saturation improved without the use of methylene blue. However, during his hospitalization, the patient also developed a pulmonary embolism and had evidence of acute coronary syndrome. Later, the patient died of multiple complications related to his cancer co-morbidity on day 12 of admission. CONCLUSIONS: Blood transfusion and supplemental oxygen, without the use of methylene blue, may be an appropriate therapeutic alternative in rasburicase-induced methemoglobinemia treatment.

Niemann-Pick C1 affects the gene delivery efficacy of degradable polymeric nanoparticles.

Despite intensive research effort, the rational design of improved nanoparticulate drug carriers remains challenging, in part due to a limited understanding of the determinants of nanoparticle entry and transport in target cells. Recent studies have shown that Niemann-Pick C1 (NPC1), the lysosome membrane protein that mediates trafficking of cholesterol in cells, is involved in the endosomal escape and subsequent infection caused by filoviruses, and that its absence promotes the retention and efficacy of lipid nanoparticles encapsulating siRNA. Here, we report that NPC1 deficiency results in dramatic reduction in internalization and transfection efficiency mediated by degradable cationic gene delivery polymers, poly(ß-amino ester)s (PBAEs). PBAEs utilized cholesterol and dynamin-dependent endocytosis pathways, and these were found to be heavily compromised in NPC1-deficient cells. In contrast, the absence of NPC1 had minor effects on DNA uptake mediated by polyethylenimine or Lipofectamine 2000. Strikingly, stable overexpression of human NPC1 in chinese hamster ovary cells was associated with enhanced gene uptake (3-fold) and transfection (10-fold) by PBAEs. These findings reveal a role of NPC1 in the regulation of endocytic mechanisms affecting nanoparticle trafficking. We hypothesize that in-depth understanding sites of entry and endosomal escape may lead to highly efficient nanotechnologies for drug delivery.

L'entrée du virus Ébola et Marburg : interaction entre la glycoprotéine virale et les facteurs cellulaires.

Ebola and Marburg viruses cause outbreaks of highly lethal infection in central Africa. In the last few years, rapid progress has been made in understanding how these viruses are transmitted and spread. These studies show that the glycoprotein GP that protrudes from the virus envelope mediates membrane fusion and infection. Activation of the GP membrane fusion activity is triggered by a multi-step pathway initiated by binding to lectins expressed on the cell surface. After uptake of lectin-bound particles by macropinocytosis, virus-containing vesicles are transported to late endosomes and lysosomes containing the protease cathepsin B and the membrane protein Niemann-Pick C1 (NPC1), which are essential for infection. Recent studies indicate that cathepsin B cleaves GP, removing heavily glycosylated sequences and exposing the domain in GP that is a ligand for NPC1. Although more studies are needed, current evidence strongly suggests that binding of protease-cleaved GP to NPC1 is the signal that activates virus membrane fusion and infection. Importantly, small molecules that target NPC1 and interfere with GP binding and ebolavirus infection have been identified. A major goal is to develop these inhibitors into anti-viral drugs.

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry.

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and "cuevaviruses") cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP(1,2), to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP(1,2), ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP(1,2)-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP(1,2), as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP(1,2)-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms.

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.

Receptor-induced thiolate couples Env activation to retrovirus fusion and infection.

According to current models of retrovirus infection, receptor binding to the surface subunit (SU) of the envelope glycoprotein (Env) triggers a conformational change in the transmembrane subunit (TM) that mediates virus fusion to cell membranes. To understand how this occurs, we investigated the role of the receptor Tva in avian leukosis virus-A (ALV-A) infection. We find that Tva binding induced the formation of a reactive thiolate on Cys38 (Cys38-S- in SU. Both chemical and genetic inactivation of Cys38-S- completely abrogated ALV fusion and infection. Remarkably, Cys38-S- does not mediate isomerization of the SU-TM disulfide bond and is not required for Tva-induced activation of TM, including pre-hairpin association with membranes and low pH assembly of helical bundles. These findings indicate that, contrary to current models, receptor activation of TM is not sufficient for ALV fusion and infection and that formation of a reactive thiolate is an additional receptor-dependent step.

Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection.

Ebola virus (EboV) causes rapidly fatal hemorrhagic fever in humans and there is currently no effective treatment. We found that the infection of African green monkey kidney (Vero) cells by vesicular stomatitis viruses bearing the EboV glycoprotein (GP) requires the activity of endosomal cysteine proteases. Using selective protease inhibitors and protease-deficient cell lines, we identified an essential role for cathepsin B (CatB) and an accessory role for cathepsin L (CatL) in EboV GP-dependent entry. Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection. CatB and CatB/CatL inhibitors diminish the multiplication of infectious EboV-Zaire in cultured cells and may merit investigation as anti-EboV drugs.

Repair of cytokine-induced DNA damage in cultured rat islets of Langerhans.

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways.

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.

Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line.

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58-65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.

The mature avian leukosis virus subgroup A envelope glycoprotein is metastable, and refolding induced by the synergistic effects of receptor binding and low pH is coupled to infection.

The spring-loaded model stipulates that influenza virus infection is coupled to the transition of the virus hemagglutinin (HA) from a metastable conformation to a highly stable conformation at low pH. The properties of retrovirus envelope glycoproteins indicate that infection is coupled to an analogous conformational change. As a test of this hypothesis, the requirements for avian leukosis virus A (ALV-A) infection were examined. These studies indicate that, like HA, the conformation of the mature ALV-A envelope glycoprotein is metastable and that infection is linked to refolding at low pH. However, unlike HA, low-pH activation is only observed after priming by receptor. Therefore, ALV-A infection is dependent on the synergistic effects of receptor binding and low pH, suggesting that receptor binding superimposes an additional constraint on activation of ALV-A fusion that proceeds by a mechanism comparable to that of influenza virus.

Visualization of retroviral replication in living cells reveals budding into multivesicular bodies.

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host-derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)-infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus-like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome-based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.