Silica-based cationic bilayers as immunoadjuvants

Background  

Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design.     

Viral Protein Determinants of Lassa Virus Entry and Release from Polarized Epithelial Cells

The epithelium plays a key role in the spread of Lassa virus. Transmission from rodents to humans occurs mainly via inhalation or ingestion of droplets, dust, or food contaminated with rodent urine. Here, we investigated Lassa virus infection in cultured epithelial cells and subsequent release of progeny viruses. We show that Lassa virus enters polarized Madin-Darby canine kidney (MDCK) cells mainly via the basolateral route, consistent with the basolateral localization of the cellular Lassa virus receptor α-dystroglycan. In contrast, progeny virus was efficiently released from the apical cell surface. Further, we determined the roles of the glycoprotein, matrix protein, and nucleoprotein in directed release of nascent virus. To do this, a virus-like-particle assay was developed in polarized MDCK cells based on the finding that, when expressed individually, both the glycoprotein GP and matrix protein Z form virus-like particles. We show that GP determines the apical release of Lassa virus from epithelial cells, presumably by recruiting the matrix protein Z to the site of virus assembly, which is in turn essential for nucleocapsid incorporation into virions.Lassa virus (LASV), a member of the family Arenaviridae, is a highly pathogenic agent causing hemorrhagic fever as a severe clinical manifestation. Arenaviruses are currently classified into more than 20 species, which are divided into the Old World and New World virus complexes (10). The Old World group includes the prototype lymphocytic choriomeningitis virus (LCMV) and the highly human-pathogenic viruses LASV and Lujo virus, as well as the nonpathogenic Ippy, Mobala, Mopeia, and Kodoko viruses (7, 21, 36). The New World virus complex contains among others, the hemorrhagic fever-associated Junín, Machupo, Guanarito, and Sabiá viruses and the recently discovered Chapare virus (14).With the exception of the New World virus Tacaribe virus, which was isolated from fruit bats, all arenaviruses have specific rodent species as their natural reservoirs. Rodents of the Mastomys natalensis species complex were identified as the natural host of LASV in certain countries in West Africa, including Sierra Leone, Nigeria, Guinea, and Liberia (26, 35, 49). An estimated 100,000 to 300,000 human LASV infections occur annually, of which approximately 30% result in illness, which can range from mild, flu-like symptoms to fulminant hemorrhagic fever with a mortality rate of about 16% of hospitalized cases (47, 48). Due to the severe or even fatal outcome of disease, unavailability of vaccine prophylaxis, and inadequate therapeutic treatment options, LASV is classified as a biosafety level 4 agent.The primary transmission route of LASV from its host to humans is by direct exposure to virus-containing urine, which may occur via the respiratory tract, through inhalation of infected particulates, or via ingestion of contaminated food (62). Moreover, hunting and preparation for consumption of rodents have also been identified as possible risk factors for rodent-to-human transmission of LASV (67). LASV is spread from human-to-human by contact with infectious body fluids or through nosocomial contaminations (22, 27). During the infection process, virus contacts the epithelial layers of the body and, after breaking through the epithelial tissue barrier, exploits dendritic cells for further dissemination (3, 15). It has been shown for LASV, as well as for other arenaviruses, that during the course of infection, infectious virus particles are released from epithelia into body fluids and urine (32, 45, 71).As epithelial layers play a pivotal role not only in initial virus infection but also in release of virus progeny during the early stages of infection, virus spread within the organism and virus release for further transmission, the polarity of entry and release from polarized epithelia has been studied extensively with various viruses. Virus entry in polarized cells is correlated with the apical or basolateral localization of the responsible virus receptor (24, 34, 68). Viruses that are transmitted through aerosols or surface contact with body fluids are generally thought to enter the epithelial barrier from the apical side, whereas virus infections due to injuries or transmission from animals'' bites and scratches enter epithelial cell layers from the basolateral side. Further, the spread of disease is also dependent on the directional release of the virus from epithelial cells. In general, basolateral virus budding is thought to cause systemic infections, whereas local infections are a result of viruses that are released predominantly from the apical side (69). Fitting with this model, budding of wild-type Sendai virus is restricted to the apical domain of polarized cells and causes a local respiratory infection, whereas systemic spread of a Sendai virus mutant could be attributed mainly to its bipolar virus release (66). The direction of entry and release can also be highly dependent on the type of tissue involved, as Sindbis and Semliki Forest viruses show differences in directed release in colon and thyroid gland cells (75). Similar differences in polarized virus release have also been shown for different members within a single virus family (59).In order to understand virus dissemination within the organism, it is of interest to determine on which side viruses enter and leave polarized epithelial cell layers. Here, we present data on directional LASV invasion into polarized MDCK cell culture and demonstrate a directional release of LASV from these cells. Furthermore, we have elucidated how Lassa virus proteins interact to direct budding and release of LASV progeny from epithelial cell layers.     

Favipiravir (T-705) Inhibits Junín Virus Infection and Reduces Mortality in a Guinea Pig Model of Argentine Hemorrhagic Fever

Background

Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses.

Methodology/Principal Findings

To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1–2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33–40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection.

Conclusions/Significance

The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶瞬时表达小鼠模型的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶体内活性评价模型。方法:利用NS4A/B是NS3/4A丝氨酸蛋白酶作用底物的特性,构建融合基因NS3/NS4A/B-SEAP,底物片段NS4A/B插在NS3/4A和人分泌性碱性磷酸酶(SEAP)之间,融合基因表达后SEAP的分泌依赖于有活性的NS3/4A在NS4A/B位点的切割。将含融合基因的质粒NS3/4A(△4AB)SEAP通过水动力转染技术转染到小鼠体内,检测小鼠血清中SEAP的活性,高活性的SEAP是该评价体系成立的证据。结果与结论:在瞬时表达NS3/4A的小鼠血清中检测到了高活性的SEAP,建立了可用于评价抗NS3/4A的小鼠体内瞬时模型。     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.      

The phytochrome gene family in grasses (Poaceae): a phylogeny and evidence that grasses have a subset of the loci found in dicot angiosperms

The phytochrome nuclear gene family encodes photoreceptor proteins that mediate developmental responses to red and far red light throughout the life of the plant. From studies of the dicot flowering plant Arabidopsis, the family has been modeled as comprising five loci, PHYA- PHYE. However, it has been shown recently that the Arabidopsis model may not completely represent some flowering plant groups because additional PHY loci related to PHYA and PHYB of Arabidopsis apparently have evolved independently several times in dicots, and monocot flowering plants may lack orthologs of PHYD and PHYE of Arabidopsis. Nonetheless, the phytochrome nucleotide data were informative in a study of organismal evolution because the loci occur as single copy sequences and appear to be evolving independently. We have continued our investigation of the phytochrome gene family in flowering plants by sampling extensively in the grass family. The phytochrome nuclear DNA data were cladistically analyzed to address the following questions: (1) Are the data consistent with a pattern of differential distribution of phytochrome genes among monocots and higher dicots, with homologs of PHYA, B, C, D, and E present in higher dicots, but of just PHYA, B, and C in monocots, and (2) what phylogenetic pattern within Poaceae do they reveal? Results of these analyses, and of Southern blot experiments, are consistent with the observation that the phytochrome gene family in grasses comprises the same subset of loci detected in other monocots. Furthermore, for studies of organismal phylogeny in the grass family, the data are shown to provide significant support for relationships that are just weakly resolved by other data sets.      

Drastic changes in lake ecosystem development as a consequence of flax retting: a multiproxy palaeolimnological study of Lake Kooraste Linajärv,Estonia

This study demonstrates the power of multiproxy palaeolimnological analyses in investigating environmental changes in the Lake Kooraste Linajärv ecosystem through historical time in response to flax retting. Flax retting history was proven by applying pollen and macrofossil evidence and by using several biotic and geochemical proxies on a sediment core. Continuous findings of flax pollen and macrofossil remains in lake sediments were considered as strong evidence for the occurrence of retting. Analyses of the well-dated sediment core show the consequences of flax retting in the lake. As a result, the once clear soft water oligotrophic endorheic lake with limited sedimentation has turned into a hypertrophic high-sedimentation lake with anoxic bottom water, strong stratification and intense water blooms. Despite the fact that flax retting was forbidden in Estonia around ad 1950s and retting has not occurred over the last six decades, anthropogenic alterations were so pervasive in the past, that they have prevented any lake water improvements until the present-day.     

Design and evaluation of pyrazolopyrimidines as KCNQ channel modulators

Effective treatments of neuropathic pain have been a focus of many discovery programs. KCNQ (kv7) are voltage gated potassium channel openers that have the potential for the treatment of CNS disorders including neuropathic pain. Clinical studies have suggested agents such as Retigabine to be a modulator of pain-like effects such as hyperalgesia and allodynia. In this paper, we describe the discovery and evaluation of a series of novel pyrazolopyrimidines and their affinity for potassium channels KCNQ2/3. These pyrazolopyrimidines have also shown good efficacy in the capsaicin-induced acute and secondary mechanical allodynia model and excellent pharmacokinetic properties, which may be superior to Retigabine.