A new genus and species of cestode (Rhinebothriidea) from Mobula kuhlii (Rajiformes: Mobulidae) from Malaysian Borneo

The cestode fauna of the shortfin devilray, Mobula kuhlii (Müller & Henle, 1841) was examined for the first time. The work resulted in the discovery of a new genus and species of rhinebothriidean tapeworm. Crassuseptum pietrafacei, n. gen. n. sp. is erected here on the basis of its unique scolex and proglottid morphology. Histological sections and examination by light and scanning electron microscopy demonstrated that this new genus differs from all other rhinebothriidean genera in its possession of bothridia in which the proximal and distal sides are confluent, i.e., not separated by a rim of tissue, and in its possession of testes that extend to the posterior margin of the ovary. This new species is characterized in part by its possession of stalked, elongate bothridia lacking lateral constrictions, with 13-15 prominent transverse bothridial septa and 4 reduced transverse septa, craspedote proglottids, each with 2-3 layers of testes in cross section, and a vas deferens that joins the cirrus sac at its anterior margin. Histological and optical sections through bothridial septa revealed that the transverse septa are formed by septal muscles separate from bothridial radial musculature, extending from the anterior side to the posterior side of each septum. This is only the second species rhinebothriidean cestode reported from mobulids. This study adds to the number of new species and genera of elasmobranch cestodes discovered off of the island of Borneo.     

NS1-Truncated Live Attenuated Virus Vaccine Provides Robust Protection to Aged Mice from Viral Challenge

Immunological changes associated with age contribute to the high rates of influenza virus morbidity and mortality in the elderly. Compounding this problem, aged individuals do not respond to vaccination as well as younger, healthy adults. Efforts to increase protection to this demographic group are of utmost importance, as the proportion of the population above the age of 65 is projected to increase in the coming decade. Using a live influenza virus with a truncated nonstructural protein 1 (NS1), we are able to stimulate cellular and humoral immune responses of aged mice comparable to levels seen in young mice. Impressively, a single vaccination provided protection following stringent lethal challenge in aged mice.     

Properties of the Adeno-Associated Virus Assembly-Activating Protein

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.     

Reconstructing the origins of high-alpine niches and cushion life form in the genus Androsace S.L. (Primulaceae)

Relatively, few species have been able to colonize extremely cold alpine environments. We investigate the role played by the cushion life form in the evolution of climatic niches in the plant genus Androsace s.l., which spreads across the mountain ranges of the Northern Hemisphere. Using robust methods that account for phylogenetic uncertainty, intraspecific variability of climatic requirements and different life-history evolution scenarios, we show that climatic niches of Androsace s.l. exhibit low phylogenetic signal and that they evolved relatively recently and punctually. Models of niche evolution fitted onto phylogenies show that the cushion life form has been a key innovation providing the opportunity to occupy extremely cold environments, thus contributing to rapid climatic niche diversification in the genus Androsace s.l. We then propose a plausible scenario for the adaptation of plants to alpine habitats.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

Uroplakins do not restrict CO2 transport through urothelium

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (～1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (～7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (～ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.     

Direct electrochemistry of novel affinity-tag immobilized recombinant horse heart cytochrome c

During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity-tag binding strategies for the directed immobilization of horse heart cytochrome c (cytc) on gold electrode surfaces to achieve this goal. Horse heart cytc was expressed in E. coli either as non-modified or genetically modified, i.e. histidine (his)-tag containing protein. The his-tags were introduced at defined positions at the N- or C-terminus of the polypeptide. It was our aim to generate tagged-versions of cytc that facilitate strong electronic coupling between protein and electrode and, at the same time, retain their catalytic and regulatory properties. The combination of different immobilization strategies, e.g. his-tag and electrostatic immobilization also opens new avenues for bivalent immobilization of proteins. This is of interest for molecular bioelectronic and biosensing applications where the proteins are immobilized between two crossing electrodes.     

Pheromones: fish fear factor

Fish, like many other animals, panic when another individual is injured. Now, the chemical nature of a substance that mediates this reaction has been uncovered.