Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-gamma-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.     

Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapiagen. nov. to accommodate the Southeast Asian species.     

Machine Learning Methods Enable Predictive Modeling of Antibody Feature:Function Relationships in RV144 Vaccinees

The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.     

Conformational propagation with prion-like characteristics in a simple model of protein folding

Protein refolding/misfolding to an alternative form plays an aetiologic role in many diseases in humans, including Alzheimer's disease, the systemic amyloidoses, and the prion diseases. Here we have discovered that such refolding can occur readily for a simple lattice model of proteins in a propagatable manner without designing for any particular alternative native state. The model uses a simple contact energy function for interactions between residues and does not consider the peculiarities of polypeptide geometry. In this model, under conditions where the normal (N) native state is marginally stable or unstable, two chains refold from the N native state to an alternative multimeric energetic minimum comprising a single refolded conformation that can then propagate itself to other protein chains. The only requirement for efficient propagation is that a two-faced mode of packing must be in the ground state as a dimer (a higher-energy state for this packing leads to less efficient propagation). For random sequences, these ground-state dimeric configurations tend to have more beta-sheet-like extended structure than almost any other sort of dimeric ground-state assembly. This implies that propagating states (such as for prions) are beta-sheet rich because the only likely propagating forms are beta-sheet rich. We examine the details of our simulations to see to what extent the observed properties of prion propagation can be predicted by a simple protein folding model. The formation of the alternative state in the present model shows several distinct features of amyloidogenesis and of prion propagation. For example, an analog of the phenomenon of conformationally distinct strains in prions is observed. We find a parallel between 'glassy' behavior in liquids and the formation of a propagatable state in proteins. This is the first report of simulation of conformational propagation using any heteropolymer model. The results imply that some (but not most) small protein sequences must maintain a sequence signal that resists refolding to propagatable alternative native states and that the ability to form such states is not limited to polypeptides (or reliant on regular hydrogen bonding per se) but can occur for other protein-like heteropolymers.     

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.     

Morphological and Glucose Metabolism Abnormalities in Alcoholic Korsakoff's Syndrome: Group Comparisons and Individual Analyses

Background

Gray matter volume studies have been limited to few brain regions of interest, and white matter and glucose metabolism have received limited research attention in Korsakoff''s syndrome (KS). Because of the lack of brain biomarkers, KS was found to be underdiagnosed in postmortem studies.

Methodology/Principal Findings

Nine consecutively selected patients with KS and 22 matched controls underwent both structural magnetic resonance imaging and ¹⁸F-fluorodeoxyglucose positron emission tomography examinations. Using a whole-brain analysis, the between-group comparisons of gray matter and white matter density and relative glucose uptake between patients with KS and controls showed the involvement of both the frontocerebellar and the Papez circuits, including morphological abnormalities in their nodes and connection tracts and probably resulting hypometabolism. The direct comparison of the regional distribution and degree of gray matter hypodensity and hypometabolism within the KS group indicated very consistent gray matter distribution of both abnormalities, with a single area of significant difference in the middle cingulate cortex showing greater hypometabolism than hypodensity. Finally, the analysis of the variability in the individual patterns of brain abnormalities within our sample of KS patients revealed that the middle cingulate cortex was the only brain region showing significant GM hypodensity and hypometabolism in each of our 9 KS patients.

Conclusions/Significance

These results indicate widespread brain abnormalities in KS including both gray and white matter damage mainly involving two brain networks, namely, the fronto-cerebellar circuit and the Papez circuit. Furthermore, our findings suggest that the middle cingulate cortex may play a key role in the pathophysiology of KS and could be considered as a potential in vivo brain biomarker.     

Transgenic tomato plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> thionin (<Emphasis Type="Italic">Thi2.1</Emphasis>) driven by fruit-inactive promoter battle against phytopathogenic attack

Tomato is one of the most important crop plants; however, attacks by pathogens can cause serious losses in production. In this report, we explore the potential of using the Arabidopsis thionin (Thi2.1) gene to genetically engineer enhanced resistance to multiple diseases in tomato. Potential thionin toxicity in fruits was negated by the use of a fruit-inactive promoter to drive the Thi2.1 gene. In transgenic lines containing RB7/Thi2.1, constitutive Thi2.1 expression was detected in roots and incidentally in leaves, but not in fruits. Disease assays revealed that the transgenic lines that were tested conferred significant levels of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW). Further studies indicated that BW disease progression in transgenic lines was delayed by a systemic suppression of bacterial multiplication. By adopting a safe genetic engineering strategy, the present investigation is another step forward demonstrating thionin practicality in crop protection.     

Structural and biochemical characterization of a quinol binding site of Escherichia coli nitrate reductase A

The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 A of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.