A compact and low-weight sputtering unit for in situ investigations of thin film growth at synchrotron radiation beamlines.

In this work, we report on a highly variable, compact, and light high-vacuum sputter deposition unit designed for in situ experiments using synchrotron radiation facilities. The chamber can be mounted at various synchrotron beamlines for scattering experiments in grazing incidence geometry. The sample position and the large exit window allow to perform x-ray experiments up to large q values. The sputtering unit is easy to mount on existing experimental setups and can be remote-controlled. In this paper, we describe in detail the design and the performance of the new sputtering chamber and present the installation of the apparatus at different 3rd generation light sources. Furthermore, we describe the different measurement options and present some selected results. The unit has been successfully commissioned and is now available for users at PETRA III at DESY.

Nanobatteries in redox-based resistive switches require extension of memristor theory.

Redox-based nanoionic resistive memory cells are one of the most promising emerging nanodevices for future information technology with applications for memory, logic and neuromorphic computing. Recently, the serendipitous discovery of the link between redox-based nanoionic-resistive memory cells and memristors and memristive devices has further intensified the research in this field. Here we show on both a theoretical and an experimental level that nanoionic-type memristive elements are inherently controlled by non-equilibrium states resulting in a nanobattery. As a result, the memristor theory must be extended to fit the observed non-zero-crossing I-V characteristics. The initial electromotive force of the nanobattery depends on the chemistry and the transport properties of the materials system but can also be introduced during redox-based nanoionic-resistive memory cell operations. The emf has a strong impact on the dynamic behaviour of nanoscale memories, and thus, its control is one of the key factors for future device development and accurate modelling.

What's behind a face: person context coding in fusiform face area as revealed by multivoxel pattern analysis.

The identification of a face comprises processing of both visual features and conceptual knowledge. Studies showing that the fusiform face area (FFA) is sensitive to face identity generally neglect this dissociation. The present study is the first that isolates conceptual face processing by using words presented in a person context instead of faces. The design consisted of 2 different conditions. In one condition, participants were presented with blocks of words related to each other at the categorical level (e.g., brands of cars, European cities). The second condition consisted of blocks of words linked to the personality features of a specific face. Both conditions were created from the same 8 × 8 word matrix, thereby controlling for visual input across conditions. Univariate statistical contrasts did not yield any significant differences between the 2 conditions in FFA. However, a machine learning classification algorithm was able to successfully learn the functional relationship between the 2 contexts and their underlying response patterns in FFA, suggesting that these activation patterns can code for different semantic contexts. These results suggest that the level of processing in FFA goes beyond facial features. This has strong implications for the debate about the role of FFA in face identification.

Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle.

Despite the potential of DNA vaccines to induce strong, balanced immune responses in small experimental species, the immune responses to DNA immunization in larger species have generally been moderate and inconsistent. In this study, the TriGridtrade mark Delivery System (TDS), an electroporation-based DNA delivery platform, was evaluated for administration of DNA vaccines to calves. When compared to conventional intramuscular delivery, TDS-based delivery markedly and consistently enhanced gene expression from a plasmid encoding a reporter gene, secreted alkaline phosphatase, and improved cell-mediated and humoral immune responses to a plasmid encoding a model antigen, hepatitis B surface antigen. Importantly, the TDS-based procedure was well tolerated by the calves, which did not need to be anesthetized or sedated. These results suggest that the TDS is a useful delivery method for DNA vaccines in cattle.

DNA prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime-protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-gamma-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-gamma production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Music Therapy Helping to Work Through Grief and Finding a Personal Identity.

This article describes a qualitative single-case study of the treatment of a woman having problems with grief and finding a personal identity. In this case the treatment has been supported by research techniques like categorizing, developing themes, writing memos, member checking, peer debriefing, and triangulation. During treatment, diagnostic themes were generated such as: problems of identity, low self-esteem, a lack of assertiveness, and problems expressing feelings and problems in relationships. These themes became important along with feelings of depression which she experienced since the death of her beloved husband. The study describes how the client was able to express, in an unconscious way, a part of her personality, which she had been suppressing since childhood, through playing the piano and vocalizing during the music therapy process. The music therapist used several techniques of improvisation to support the client's cautious steps into a new musical and a new personal world. Tables of music therapy improvisations show how the client became musically expressive, how she found a personal melody and how she developed a closer relationship with the music therapist through musical interaction. Guidelines for similar cases and generated hypotheses about the contribution of music are enclosed as results of research.

Monoclonal antibodies to the E2 protein of a new genotype (type 2) of bovine viral diarrhea virus define three antigenic domains involved in neutralization.

Bovine viral diarrhea virus (BVDV) has recently been segregated into two genotypes, namely, BVDV 1 and BVDV 2. Viruses of the BVDV 2 genotype are a cause of hemorrhagic and acute fatal disease in cattle in the US and Canada. In this study, monoclonal antibodies (mAbs) to the newly described BVDV 2 were produced after immunization with virus or a combination of virus and E2 peptide. From an original panel of 17 mAbs, 13 mAbs were identified as E2-specific by reactivity with a BVDV 2 recombinant E2 protein expressed in insect cells. Nine E2 mAbs were observed to be virus-neutralizing. The E2 epitopes represented by the mAbs were found to be highly conserved among BVDV 2 isolates associated with hemorrhagic or severe disease in cattle. Except for one virus-neutralizing E2 mAb, the mAbs showed few or relatively weak cross-reactions with BVDV 1. Two non-neutralizing E2 mAbs were BVDV 2-specific. In contrast to BVDV 1 for which conserved neutralizing epitopes have been mapped in one immunodominant domain, the virus-neutralizing E2 mAbs produced to BVDV 2 were found to bind to highly conserved epitopes in three antigenic domains.

Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants.

Bovine viral diarrhoea viruses (BVDV) have recently been segregated into two genotypes, BVDV 1 and BVDV 2. However, the antigenic differences and similarities of BVDV 1 and BVDV 2 remain poorly defined. In this study, the E2 epitopes of two neutralizing monoclonal antibodies (mAbs) produced against an isolate of BVDV 1 were mapped. The mAb 157, previously determined to be broadly cross-reactive to BVDV, was discovered to be BVDV 1-specific, whereas mAb 348 bound to and neutralized BVDV 2. Both mAbs bound to epitopes within the first 192 amino acids of the E2 protein as determined by reactions with a C-terminally truncated E2. To identify critical amino acids affecting these epitopes, mAb escape mutants were selected for sequencing from BVDV 1 and BVDV 2 strains with different (wild-type) mAb binding phenotypes. In addition, the E2 gene of several BVDV were sequenced and the sequences were compared with amino acid changes in mutant viruses. Single nucleotide changes in escape mutants selected with mAb 157 resulted in deduced amino acid changes at E2 positions 9, 32 or 72. Amino acid changes at position 72 also affected the epitope of mAb 348. Alignment of E2 nucleotide sequences revealed that BVDV 2 are missing six nucleotides encoding the equivalent of amino acids 31 and 32 of BVDV 1 and thus, this difference can account for the BVDV 1-specificity of mAb 157. Single nucleotide mutations in mAb 348 escape mutants of BVDV 1 and BVDV 2 resulted in changes in 3 amino acids in the previously described immunodominant 71-74 region (Virology 190, 763-772). A fourth amino acid change observed in a mutant of BVDV 2 extended this region to position 77. Thus, the amino acid changes affecting the conserved epitope of mAb 348 occurred in a short spatial array over only seven amino acids, unlike the described composite epitopes previously mapped to this region.

Characterization of the interaction between VP8 of bovine rotavirus C486 and cellular components on MA-104 cells and erythrocytes.

Rotavirus VP8*, the N-terminal trypsin cleavage product of VP4, has been shown to bind to MA-104 cells and human O type erythrocytes. To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E. coli and radiolabelled with 35S-methionine. The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV. To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8*. rVP8* showed HA which could be inhibited by antiserum to BRV. This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction. To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used. First, a peptide spanning aa 230-247 was synthesized and antisera was raised against the peptide to see whether it could inhibit HA of rVP8*. Second, a truncated form of VP8* (tVP8*: aa 1-229) was expressed to examine its hemagglutinating activity. Third, the dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE. The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA. To characterize the interaction between the HA domain and sialic acid receptors, erythrocytes were treated with sialidases of different specificities. Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary.

Functional characterization of bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins expressed by adenovirus recombinants.

We constructed replication-competent human adenovirus type 5 (HAd5) recombinants (HAd5-HN and HAd5-F) containing the bovine parainfluenza virus type 3 (BPIV3) hemagglutinin-neuraminidase (HN) or fusion (F) gene under the control of the simian virus 40 (SV40) regulatory sequences. These genes were inserted in the early region 3 (E3) of the HAd5 genome in the E3 parallel orientation. Expression of HN or F in HAd5-HN- or HAd5-F-infected cell extracts, respectively, was observed by immunoprecipitation using a BPIV3-specific polyclonal antiserum. Our results suggest that HN and F expressed by HAd5 recombinants were functionally similar to the native HN and F expressed in BPIV3-infected cells.

Mouse choroideremia gene mutation causes photoreceptor cell degeneration and is not transmitted through the female germline.

Choroideremia (CHM) is an X-linked progressive eye disorder which results from defects in the human Rab escort protein-1 (REP-1) gene. A gene targeting approach was used to disrupt the mouse chm/rep-1 gene. Chimeric males transmitted the mutated gene to their carrier daughters but, surprisingly, these heterozygous females had neither affected male nor carrier female offspring. The targeted rep-1 allele was detectable, however, in male as well as female blastocyst stage embryos isolated from a heterozygous mother. Thus, disruption of the rep-1 gene gives rise to lethality in male embryos; in female embryos it is only lethal if the mutation is of maternal origin. This observation can be explained by preferential inactivation of the paternal X chromosome in murine extraembryonic membranes suggesting that expression of the rep-1 gene is essential in these tissues. In both heterozygous females and chimeras the rep-1 mutation causes photoreceptor cell degeneration. Consequently, conditional rescue of the embryonic lethal phenotype of the rep-1 mutation may provide a faithful mouse model for choroideremia.

Protective immunity in cattle following vaccination with conventional and marker bovine herpesvirus-1 (BHV1) vaccines.

The efficacy of two experimental subunit gD vaccines and two commercial whole virus vaccines was determined in a bovine herpesvirus-1 (BHV1) challenge trial. Full-length gD and a truncated, secreted form of gD (tgD) were produced using a vaccinia virus expression system and purified by affinity chromatography. Comparison of these forms of gD did not reveal significant structural or antigenic differences. Calves immunized with gD or tgD in avridine developed significantly (P < 0.05) higher neutralizing antibody titers in the serum and nasal mucosa than animals vaccinated with killed virus (KV) or modified live virus (MLV). Following challenge with BHV1, all vaccinated calves had significantly (P < 0.05) lower rectal temperatures and clinical scores than those in the placebo group. In contrast to the KV-, MLV- and placebo-vaccinated calves, the gD and tgD-immunized animals experienced minimal weight loss and virus shedding post-challenge. Glycoprotein B-specific antibodies were detected in KV- and MLV-vaccinated calves, but not in gD- or tgD-immunized animals. These data suggest that full-length or truncated gD, when formulated in an appropriate adjuvant, is more effective than two KV and MLV vaccines and may be used as a marker vaccine for concurrent vaccination and eradication programs of BHV1.

Performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single Saskatchewan beef herd.

Fifty-one calves from 652 cows and heifers that calved on a Saskatchewan ranch in 1992 were identified as persistently infected with bovine viral diarrhea virus (BVDV), based on virological and necropsy findings. Herd records suggested a further 20 calves that died between birth and weaning were probably also persistently infected. Subsequent to weaning, all surviving persistently infected calves were transferred to one pen in a 10,000 head commercial feedlot, to mimic normal management practice in western Canadian beef herds. On average, when compared with healthy, BVDV-negative herdmates, persistently infected calves were "poor doers" and had poor survivability, with only 4 persistently infected calves surviving to 1 year of age. There was no difference (P > 0.05) in survival between male and female persistently infected calves. The clinical, pathological, and virological findings from these persistently infected calves varied over time. The majority of persistently infected calves had gross pathological lesions at necropsy, consistent with mucosal disease. However, approximately 25% of the persistently infected calves had gross pneumonic lesions at necropsy, with no or only mild lesions of mucosal disease. A wide variety of other lesions were also noted in persistently infected calves at necropsy. Therefore, the possibility that BVDV-induced lesions can be misdiagnosed is very real. The results of this study indicate that persistent infection with BVDV should always be considered in calves with chronic ill thrift, chronic enteritis, or respiratory disease.

Molecular basis of choroideremia (CHM): mutations involving the Rab escort protein-1 (REP-1) gene.

Choroideremia (CHM) is an X-linked recessive eye disease that results from mutations involving the Rab escort protein-1 (REP-1) gene. In 18 patients deletions of different sizes have been found. Two females suffering from CHM were reported to have translocations that disrupt the REP-1 gene. In 22 patients, small mutations have been identified. Interestingly, these are all nonsense, frameshift or splice-site mutations; with one possible exception, missense mutations have not been found. This comprises all the known mutations in the disease.

Characterization of the haemorrhagic enteritis virus genome and the sequence of the putative penton base and core protein genes.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0%, 53.0% and 44.2% similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3-48.6 m.u. on the HEV genome and had a remarkably low G+C content (33.8%). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

Immunodominant E2 (gp53) sequences of highly virulent bovine viral diarrhea group II viruses indicate a close resemblance to a subgroup of border disease viruses.

The genes for the E2 envelope protein, which elicits virus-neutralizing antibodies, from members of the newly described group II of bovine viral diarrhea viruses (BVDVs) were cloned and sequenced. These BVDVs included a thrombocytopenic strain from the United States, a fetal bovine serum contaminant, a strain from Western Canada, and two highly virulent strains, causing high mortality rates, from Quebec. The nucleotide and amino acid sequences of these E2s had only a 60-65% homology with group I BVDV E2s but >90% homology with the E2 of a subgroup of sheep border disease viruses. The E2 gene of the NADL strain was expressed and monospecific antibodies were raised in calves and rabbits. The virus-neutralizing titers of these antisera were 15- to 80-fold lower for the heterologous group of BVDVs as compared to those for the homologous BVDVs.

The prevalence of bovine viral diarrhea virus infection in a population of feedlot calves in western Canada.

The prevalence of bovine viral diarrhea virus (BVDV) infection was examined in a population of 5129 recently weaned steer calves entering a large feedlot in central Saskatchewan from September to December 1991. Serum samples were collected within 24 h of arrival at the feedlot from every fifth calf processed and again 96 d postarrival. A microtiter virus isolation test was used to determine the prevalence of calves viremic with BVDV on entry to the feedlot. An enzyme-linked immunosorbent assay (ELISA) which detects antibody against glycoprotein 53 of the BVDV was used on paired sera to determine the seroconversion risk during the first 96 d in the feedlot. A virus neutralization (VN) test for BVDV was conducted on a sub-sample of paired sera to measure agreement in determination of seroconversion risk with the ELISA. A polymerase chain reaction (PCR) test which detects BVDV was used to determine if cattle were acutely viremic when treated for disease. The estimated prevalence of persistently infected calves in this population was < 0.1%. The seroconversion risk for BVDV was 27% (236/864) according to the ELISA and it varied from 0 to 63% among the 20 pens sampled. According to the VN test, the seroconversion risk for BVDV was 40% (132/327) and it varied from 0 to 100% among the 11 pens tested. The agreement between the ELISA and VN tests in seroconversion risk to BVDV was very poor (kappa = 0.15 +/- 0.039 SE). The prevalence of acute viremia in calves treated at the feedlot hospital was low at 4% (6/149).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the major tegument protein VP8 of bovine herpesvirus-1 in infection and immunity.

The tegument of bovine herpesvirus-1 (BHV-1) carries an abundant protein of 96 kDa, termed VP8. Immunolabeling using VP8-specific antiserum and colloidal gold-labeled protein A as the electron-dense marker was used to identify VP8 in the virions and virus-infected cells. VP8 was confirmed to be a tegument protein that, like the herpes simplex virus-1 homologue VP13/14, contains O-linked carbohydrates. VP8 was found in the nucleus of virus-infected cells as early as 2 hr postinfection. Since VP8 is a gamma2 protein, this protein cannot be newly synthesized at this time and must be acquired from the inoculum. This supports the hypothesis that early during infection, VP8 has a function in modulation of alpha gene expression. Later during infection, VP8 was observed in the cytoplasm around nucleocapsids and in dense inclusions, which accumulated in the cisternae of the Golgi. In addition, de novo-synthesized VP8 continued to accumulate in the nucleus in dense areas and around nucleocapsids. In calves, VP8 stimulated T cell proliferation and antibody production, both after BHV-1 challenge and after immunization with purified VP8. These results suggest a role for VP8 in the induction of humoral and specifically cell-mediated immunity to BHV-1.