Bacterial septicemia and herpesvirus infection in Antarctic fur seals (Arctocephalus gazella) stranded in the São Paulo coast, Brazil.

In August 2021, two juvenile male Antarctic fur seals (Arctocephalus gazella) stranded in the southeastern Brazilian coast and were referred to rehabilitation centers. The animals presented increased body temperature, prostration, respiratory distress and despite treatment died. A necropsy following a standardized protocol was performed, and formalin-fixed tissues were processed for microscopic examination. Samples were screened for morbillivirus, herpesvirus, and Brucella spp. by molecular analyses (PCR, RT-PCR). Bacteriological culture was performed in samples collected from the lungs, trachea, and lymph nodes of both cases. The main histopathologic findings were of infectious nature, including multifocal necrotizing and fibrinous mixed interstitial pneumonia, bronchiolitis, and bronchitis, with intralesional myriad bacteria associated with vascular fibrinoid necrosis. Pseudomonas aeruginosa was isolated from tracheal and lung swabs of Case 1, and Klebsiella oxytoca was found in nostril swabs, tracheobronchial lymph nodes, and lung of Case 2. Gammaherpesvirus infection was detected in both cases, and the sequences retrieved were classified into the genus Percavirus. All tested samples were PCR-negative for Brucella spp. and morbillivirus. We hypothesize that the deficient immunological status in association with starvation predisposed the reactivation of herpesvirus and secondary bacterial co-infections. To the authors' knowledge, this is the first molecular detection of herpesvirus in an Antarctic pinniped. These findings reinforce that Otariid gammaherpesvirus circulating in the Southern Hemisphere are likely endemic in the Arctocephalus genus. This report contributes to the current knowledge of health aspects affecting wild pinnipeds, especially in the poorly studied Antarctic species.

Nematic films at chemically structured surfaces.

We investigate theoretically the morphology of a thin nematic film adsorbed at flat substrate patterned by stripes with alternating aligning properties, normal and tangential respectively. We construct a simple 'exactly-solvable' effective interfacial model where the liquid crystal distortions are accounted for via an effective interface potential. We find that chemically patterned substrates can strongly deform the nematic-air interface. The amplitude of this substrate-induced undulations increases with decreasing average film thickness and with increasing surface pattern pitch. We find a regime where the interfacial deformation may be described in terms of a material-independent universal scaling function. Surprisingly, the predictions of the effective interfacial model agree semi-quantitatively with the results of the numerical solution of a full model based on the Landau-de Gennes theory coupled to a square-gradient phase field free energy functional for a two phase system.

Nematic droplets on fibers.

The emergence of new techniques for the fabrication of nematic droplets with nontrivial topology provides new routes for the assembly of responsive devices. Here we explore some of the properties of nematic droplets on fibers, which constitute the basic units of a type of device that is able to respond to external stimuli, including the detection of gases. We perform a numerical study of spherical nematic droplets on fibers. We analyze the equilibrium textures for homogeneous and hybrid boundary conditions and find that in some cases the nematic avoids the nucleation of topological defects, which would provide a different optical response. We consider in detail a homeotropic nematic droplet wrapped around a fiber with planar anchoring. We investigate the effect of an electric field on the texture of this droplet. In the presence of a dc field, the system undergoes an orientational transition above a given threshold E(c), for which a ring defect is transformed into a figure-eight defect. We also consider ac fields, at high and low frequencies, and find that the textures are similar to those observed for static fields, in contrast with recently reported experiments.

Particle selection through topographic templates in nematic colloids.

Liquid crystal colloids have been proposed as suitable candidates for responsive photonic crystals. Large scale growth of such colloidal systems is, however, a challenge and recently template-assisted assembly has been proposed to guide the growth of colloidal crystals, with controlled symmetries, in nematic liquid crystals. Known for their long-range anisotropic interactions, these colloidal systems are stabilized typically at the center of the cells due to strong particle-wall repulsion from the confining substrates. This behaviour is dramatically changed in the presence of topographic patterning. Here we propose the use of topographic modulation of surfaces to select and localize particles in nematic colloids. By considering convex and concave deformations of one of the confining surfaces we show that the colloid-flat surface repulsion may be enhanced or switched into an attraction. In particular, we find that when the colloidal particles have the same anchoring conditions as the patterned surfaces, they are strongly attracted to concave dimples, while if they exhibit different anchoring conditions they are pinned at the top of convex protrusions. Although dominated by elastic interactions the first mechanism is reminiscent of the depletion induced attraction or of the key-lock mechanism, while the second is specific to liquid crystal colloids. These long-ranged, highly tunable, surface-colloid interactions contribute to the development of template-assisted assembly of large colloidal crystals, with well defined symmetries, as required for applications.

Nematic wetting and filling of crenellated surfaces.

We investigate nematic wetting and filling transitions of crenellated surfaces (rectangular gratings) by numerical minimization of the Landau-de Gennes free energy as a function of the anchoring strength, for a wide range of the surface geometrical parameters: depth, width, and separation of the crenels. We have found a rich phase behavior that depends in detail on the combination of the surface parameters. By comparison to simple fluids, which undergo a continuous filling or unbending transition, where the surface changes from a dry to a filled state, followed by a wetting or unbinding transition, where the thickness of the adsorbed fluid becomes macroscopic and the interface unbinds from the surface, nematics at crenellated surfaces reveal an intriguingly rich behavior: in shallow crenels only wetting is observed, while in deep crenels, only filling transitions occur; for intermediate surface geometrical parameters, a new class of filled states is found, characterized by bent isotropic-nematic interfaces, which persist for surfaces structured on large scales, compared to the nematic correlation length. The global phase diagram displays two wet and four filled states, all separated by first-order transitions. For crenels in the intermediate regime re-entrant filling transitions driven by the anchoring strength are observed.

Filling and wetting transitions of nematic liquid crystals on sinusoidal substrates.

Close to sinusoidal substrates, simple fluids may undergo a filling transition, in which the fluid passes from a dry to a filled state, where the interface remains unbent but bound to the substrate. Increasing the surface field, the interface unbinds and a wetting transition occurs. We show that this double-transition sequence may be strongly modified in the case of ordered fluids, such as nematic liquid crystals. Depending on the preferred orientation of the nematic molecules at the structured substrate and at the isotropic-nematic interface, the filling transition may not exist, and the fluid passes directly from a dry to a complete-wet state, with the interface far from the substrate. More interestingly, in other situations, the complete wetting transition may be prevented, and the fluid passes from a dry to a filled state, and remains in this configuration, with the interface always attached to the substrate, even for very large surface fields. Both transitions are observed only for a same substrate in a narrow range of amplitudes.

Modeling dipolar and quadrupolar defect structures generated by chiral islands in freely suspended liquid crystal films.

We report a detailed theoretical analysis of quadrupolar interactions observed between islands, which are disklike inclusions of extra layers, floating in thin, freely suspended smectic- C liquid crystal films. Strong tangential anchoring at the island boundaries results in a strength +1 chiral defect in each island and a companion -1 defect in the film--these forming a topological dipole. While islands of the same handedness form linear chains with the topological dipoles pointing in the same direction, as reported in the literature, islands with different handedness form compact quadrupolar structures with the associated dipoles pointing in opposite directions. The interaction between such heterochiral-island-defect pairs is complex, with the defects moving to minimize the director field distortion as the distance between the islands changes. The details of the interisland potential and the trajectories of the -1 defects depend strongly on the elastic anisotropy of the liquid crystal, which can be modified in the experiments by varying the material chirality of the liquid crystal. A Landau model that describes the energetics of freely mobile defects is solved numerically to find equilibrium configurations for a wide range of parameters.

An accelerated vaccine schedule with a poly-antigenic hepatitis C virus MVA-based candidate vaccine induces potent, long lasting and in vivo cross-reactive T cell responses.

We designed and evaluated in HLA-class I transgenic mouse models a hepatitis C virus (HCV) T cell-based MVA vectored vaccine expressing three viral antigens known to be targets of potent CD8+- and CD4+-mediated responses. An accelerated (3 week-based) vaccination induced specific CD8+ T cells harboring two effector functions (cytolytic activity - both in vitro and in vivo- and production of IFNgamma) as well as specific CD4+ T cells recognizing all three vaccine antigens. Responses were long lasting (6 months), boostable by a fourth MVA vaccination and in vivo cross-reactive as demonstrated in a surrogate Listeria-based challenge assay. This candidate vaccine has now moved into clinical trials.

Elliptical soft colloids in smectic-C films.

We investigate theoretically the elliptical shapes of soft colloids in freely standing smectic- C films, that have been reported recently. The colloids favor parallel alignment of the liquid crystal molecules at their surfaces and, for sufficiently strong anchoring, will generate a pair of defects at the poles of the colloidal particles. The elastic free energy of the liquid crystal matrix will, in turn, affect the shape of the colloids. In this study we will focus on elliptical soft colloids and determine how their equilibrium shapes depend on the elastic constants of the liquid crystal, the anchoring strength, the surface tension, and the size of the colloids. A shape diagram is obtained analytically, by minimizing the Frank elastic free energy, in the limit of small eccentricities. The analytical results are verified, and generalized to arbitrary eccentricities, by numerical minimization of an appropriate Landau free energy. The latter is required for an adequate description of the topological defects when the liquid crystal correlation length is comparable to the size of the colloidal particles.

Key-lock mechanism in nematic colloidal dispersions.

We consider the interaction between two-dimensional nematic colloids and planar or sculpted walls. The elastic interaction between colloidal disks and flat walls, with homeotropic boundary conditions, is always repulsive. These repulsions may be turned into strong attractions at structured or sculpted walls, with cavities that match closely the shape and size of the colloids. This key-lock mechanism is analyzed in detail for colloidal disks and spherocylindrical cavities of various length to depth ratios, by minimizing the Landau-de Gennes free energy functional of the nematic orientational order parameter. We find that the attractions occur only for walls with cavities within a small range of the colloidal size and a narrow range of orientations with respect to the cavity's symmetry axis.

Colloidal interactions in two-dimensional nematics.

The interaction between two disks immersed in a 2D nematic is investigated i) analytically using the tensor order parameter formalism for the nematic configuration around isolated disks and ii) numerically using finite-element methods with adaptive meshing to minimize the corresponding Landau-de Gennes free energy. For strong homeotropic anchoring, each disk generates a pair of defects with one-half topological charge responsible for the 2D quadrupolar interaction between the disks at large distances. At short distance, the position of the defects may change, leading to unexpected complex interactions with the quadrupolar repulsive interactions becoming attractive. This short-range attraction in all directions is still anisotropic. As the distance between the disks decreases, their preferred relative orientation with respect to the far-field nematic director changes from oblique to perpendicular.

Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids.

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriP sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression. oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.

Identical cis-acting elements and related trans-acting factors control activity of nonviral promoter in Schizosaccharomyces pombe and mammalian cells.

We have analyzed the transcriptional activity of the human plasminogen activator inhibitor-1 promoter in the fission yeast Schizosaccharomyces pombe. This promoter is active in S. pombe, and the initiation site of transcription corresponds to the site identified previously in mammalian cells. Mutations in the AP-1-binding site (PAI-1 A box) or the HLTF-binding site (the B box), which reduced the basal and phorbol ester-induced levels of PAI-1 expression in human cells, also decreased the transcriptional activity in S. pombe. Gel retardation assays showed that an S. pombe protein binds specifically to this B box element and displays the same B box sequence requirement as HLTF. Furthermore, this yeast protein binds specifically to other HLTF-binding sites in the human immunodeficiency virus-1 long terminal repeat (LTR) and the simian virus 40 (SV40) enhancer. The B box (but not a mutated B box) strongly stimulated transcription when combined with adh downstream promoter elements, indicating that the S. pombe B box-binding protein, like HLTF, is a transcriptional activator. We conclude that the transcriptional activity of the nonviral PAI-1 promoter is controlled by the same promoter elements in S. pombe as in mammalian cells. In addition, mammalian trans-acting factors that bind to these promoter elements were shown to have counterparts with conserved DNA-binding activity in S. pombe. These results further illustrate the conservation of the mechanism of transcription between mammalian cells and fission yeast.

Characterization of upstream activating sequences involved in activation and regulation of pho4 expression in Schizosaccharomyces pombe.

In this study, a short region of the pho4 promoter located just upstream of the TATA box, called the upstream activating sequence (UAS), was shown to be responsible for both activation and regulation of pho4 expression in Schizosaccharomyces pombe. This cis-acting sequence is able to activate transcription when placed upstream of the minimal promoter of the constitutively expressed adh gene, and to ensure regulation by thiamine. The organisation of the pho4 promoter is remarkable in that UAS and TATA box are very closely associated. This proximity appears to be essential for efficient transcriptional regulation. Indeed, only slight variations in transcript levels were observed under derepression conditions when the UAS was moved away from the TATA box, while regulation of transcript levels appeared to be strongly affected. This observation suggests the existence of a negative regulatory element whose action requires very close association of UAS and TATA box. Surprisingly, when UAS was moved about 40 bp away from the adh TATA box, residual but very significant repression of gene expression by thiamine, which apparently does not result from transcriptional regulation, was revealed by measuring protein production. These data prompted us to hypothesize the existence of two distinct and cooperative mechanisms of regulation involving specific factor binding to the UAS, the first one controlling transcript levels and the second acting posttranscriptionally.