The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin.     

Staff Nurses’ Perceptions and Experiences about Structural Empowerment: A Qualitative Phenomenological Study

The aim of the study reported in this article was to investigate staff nurses’ perceptions and experiences about structural empowerment and perceptions regarding the extent to which structural empowerment supports safe quality patient care. To address the complex needs of patients, staff nurse involvement in clinical and organizational decision-making processes within interdisciplinary care settings is crucial. A qualitative study was conducted using individual semi-structured interviews of 11 staff nurses assigned to medical or surgical units in a 600-bed university hospital in Belgium. During the study period, the hospital was going through an organizational transformation process to move from a classic hierarchical and departmental organizational structure to one that was flat and interdisciplinary. Staff nurses reported experiencing structural empowerment and they were willing to be involved in decision-making processes primarily about patient care within the context of their practice unit. However, participants were not always fully aware of the challenges and the effect of empowerment on their daily practice, the quality of care and patient safety. Ongoing hospital change initiatives supported staff nurses’ involvement in decision-making processes for certain matters but for some decisions, a classic hierarchical and departmental process still remained. Nurses perceived relatively high work demands and at times viewed empowerment as presenting additional. Staff nurses recognized the opportunities structural empowerment provided within their daily practice. Nurse managers and unit climate were seen as crucial for success while lack of time and perceived work demands were viewed as barriers to empowerment.     

Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium

Summary An electrical fusion method has been used to form somatic hybrids between protoplasts of two mutant cell lines of sycamore tissue culture cells. Both mutants will not grow in a hypoxanthine-aminopterin-thymidine (HAT) medium. It was possible to select the fused hybrids from homospecific fusion products and nonfused protoplasts by the use of HAT medium. In this way the viability and regeneration of the fused cells during the first few weeks of culture could be evaluated. An electron microscopic examination of the fusion process showed that it occurred at a series of points along the surface of the plasmalemma. Cytoplasmic bridges between the two cells were formed separated by vesicles which later dispersed to give complete cytoplasmic continuity between the cells.     

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.     

The effects of various nutritional supplements on the growth,migration and differentiation ofxenopus laevis neural crest cells in vitro

Summary This study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested for cell migration and adhesion. This work was supported by establishment and major equipment grants from the Alberta Heritage Foundation for Medical Research to N. C. M. Nadine C. Milos is a Heritage Medical Research Scholar of the Alberta Heritage Foundation for Medical Research.     

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.