Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates.

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

The foundations of the working alliance in assertive community treatment teams.

BACKGROUND: In this study, we aimed to identify and define the fundamental components of the working alliance in multidisciplinary (Flexible) Assertive Community Treatment teams with shared caseloads, in order to support their daily practice and further research. METHODS: After reviewing the literature, concept mapping with professionals and clients was used to define the working alliance in (F) ACT teams. The resulting concept maps formed the basis for the working alliance assessment instrument, which was pilot tested with professionals and clients through cognitive interviews with a think-aloud procedure. RESULTS: The study led to the development of a twenty five-item assessment instrument to evaluate working alliances in multidisciplinary teams (WAM) that was comprised of three subscales: bond, task/goal and team. Two different versions were developed for clients and professionals. CONCLUSIONS: The WAM instrument was developed to determine the quality of the working alliance in (F) ACT teams. Future research will focus on testing its psychometric properties and predictive value.

Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Long-term prognostic value of preoperative anemia in patients with colorectal cancer: A systematic review and meta-analysis.

OBJECTIVE: To evaluate the long-term prognostic factor of preoperative anemia in colorectal cancer patients. BACKGROUND: Anemia is frequently observed in colorectal cancer patients, with a case incidence of 30 to 67 percent. Besides an indicator of tumor-induced blood loss and inflammation, anemia in cancer is also suggested to be a cause of inferior outcome, possibly via worsening of tumor hypoxia. As surgery is likely to enhance anemia, the long-term prognostic value of preoperative anemia seems most interesting. METHODS: Comprehensive searches were carried out in all relevant databases, including MEDLINE, Embase and Web-of-Science. To include studies addressing overall survival, follow-up had to be at least 24 months or till death. For pooling of survival results, a mixed-linear (fixed-effects) model was fit to the reported hazard ratios (HRs) to calculate a pooled estimate and confidence interval. RESULTS: We included 12 studies comprising 3588 patients to estimate the association between preoperative anemia and overall survival (OS) and disease-free survival (DFS). In a fixed-effects meta-analysis of eight studies, including both colon and rectal cancer, preoperative anemia was significantly associated with poor OS (HR 1.56; 95% CI 1.30 to 1.88; p < 0.001). A meta-analysis of seven studies also showed that preoperative anemia was significantly associated with poor DFS (HR 1.34; 95% CI 1.11 to 1.61; p = 0.002). Restricted to studies exclusively on colon cancer or rectal cancer, HRs for OS were 1.25 (95% CI 1.00 to 1.55; p = 0.05) and 2.59 (95% CI 1.68 to 4.01; p < 0.001), respectively, while HRs for DFS were 1.21 (95% CI 0.96 to 1.52; p = 0.11) and 1.61 (95% CI 1.18 to 2.21; p = 0.003). CONCLUSION: The present meta-analysis reveals that preoperative anemia is significantly associated with decreased long-term OS and DFS in rectal cancer, but not in colon cancer patients, although this meta-analysis is mainly based on retrospective studies with high heterogeneity. These results justify raised awareness about the impact of preoperative anemia on long-term survival.

Pirandello's analogy: a source for a better understanding of the social impact of stroke.

BACKGROUND: Suffering a stroke has major implications for the patient. To understand human suffering, one should understand society. Pirandello described society as a higher entity than the individual, thereby justifying human adaptability to society. We explore a qualitative finding that suggests that social trends may influence how stroke patients prioritize aspects of their rehabilitation. METHODS: We compare a contemporary patient's experience of stroke recovery with that of a fictional character from the works of Luigi Pirandello. Both patients had two main residual symptoms: hemiparesis and aphasia. RESULTS: The rehabilitation priorities of the two patients differed, and appeared to reflect the contemporaneous demands of society. Mobility was prioritized in 1910; communication was prioritized in 2010. However, essential aspects of 'being a stroke patient' remained unchanged; both patients retained a sense of self and both coped emotionally by being hopeful. CONCLUSIONS: We conclude that stroke patients respond to society's contemporaneous demands and expectations. Currently, society demands participation in a large social environment and this is reflected in stroke patients' priorities. This analogy could enable medical professionals to better understand the social impact of stroke, and consequently offer appropriate interventions to improve rehabilitation outcomes for individual patients.

Glial and neuronal proteins in serum predict outcome after severe traumatic brain injury.

OBJECTIVE: To study the ability of glial (glial fibrillary acidic protein [GFAP] and S100b) and neuronal (neuron specific enolase [NSE]) protein levels in peripheral blood to predict outcome after severe traumatic brain injury. METHODS: Eighty-five patients with severe traumatic brain injury (admission Glasgow Coma Score [GCS] < or = 8) were included. Blood samples taken at the time of hospital admission were analyzed for S100b, GFAP, and NSE. Data collected included demographic and clinical variables. Outcome was assessed using the Glasgow Outcome Scale (GOS) at 6 months post injury. RESULTS: The median serum levels of S100b, GFAP, and NSE were raised 18.3 fold (S100b), 4.6 fold (GFAP), and twofold (NSE) compared to normal reference values. S100b, GFAP, and NSE serum levels correlated significantly with the injury severity score and CT findings but not with age, sex, or GCS. S100b, GFAP, and NSE levels were significantly higher in patients who died or had a poor outcome 6 months post injury than in those who were alive or had good outcome. S100b level >1.13 microg/L was the strongest predictor of death with 100% discrimination, but GFAP (>1.5 microg/L) and NSE (>21.7 microg/L) levels also strongly predicted death (adjusted odds ratios 5.82 [for GFAP] and 3.91 [for NSE]). S100b, GFAP, and NSE all strongly predicted poor outcome (adjusted odds ratios 5.12 [S100b], 8.82 [GFAP], and 3.95 [NSE]). CONCLUSIONS: These results suggest that determination of serum levels of glial and neuronal proteins may add to the clinical assessment of the primary damage and prediction of outcome after severe traumatic brain injury.

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Moderate alcohol consumption and postprandial plasma lipids in men with different risks for coronary heart disease.

BACKGROUND: Moderate alcohol consumption is associated with a reduced coronary heart disease (CHD) risk. Epidemiologic studies have provided conflicting data which suggests that CHD protection may be modulated or may not be modulated by a person's CHD risk profile. METHODS: We examined the effects of moderate alcohol consumption (35 g/day) on postprandial lipoprotein metabolism in two groups of healthy middle-aged men who had different plasma total cholesterol, triglyceride concentrations, and body mass index (BMI), which are three major risk factors for CHD; 11 men had lower plasma lipids and BMI (L-men) and 11 men had higher plasma lipids and BMI (H-men). The effects of alcohol on postprandial lipoprotein metabolism were studied in a crossover design after an acute moderate alcohol intake both after a period of abstinence (alcohol-free beer) and after a period of moderate alcohol consumption (alcohol containing beer). RESULTS: Moderate alcohol consumption changed plasma total cholesterol, total triglycerides, and HDL composition in the postprandial period. Alcohol-induced changes were essentially the same over time in both L-men and H-men. However, changes occurred at a different overall plasma concentration for total cholesterol and total triglycerides. Also, the postprandial response to an acute moderate alcohol dose after a period of abstinence seemed not to essentially differ from the response to an acute moderate alcohol dose after a 4-week period of moderate alcohol consumption. CONCLUSIONS: These results suggest that men who differ in risk for CHD, based on plasma lipids and BMI, but without previous or underlying disease, have a similar postprandial lipid response to a moderate dose of alcohol.

Central Australian Nurse Management Model (CAN Model): a strategic approach to the recruitment and retention of remote-area nurses.

This paper introduces a new strategic approach, the Central Australian Nurse Management Model (CAN Model), to manage remote area nursing services. Central Australia is home to approximately 45,000 people, of whom 30% are Aborigines with a health status that is markedly lower than the rest of the population. While the Federal, State and Territory governments have policies in place to address health inequities, improvement has been hindered by the difficulty in recruiting and retaining suitable nursing staff in remote areas. Implementation of the three key initiatives that comprise the CAN Model has succeeded in attracting, stabilising and skilling a remote area nursing workforce, fundamental to achieving better health outcomes in Aboriginal populations.

Single-site manipulation of tomato chromosomes in vitro and in vivo using Cre-lox site-specific recombination.

With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 bp specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.

Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning.

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This 'transposition-deletion' system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.

Differential repair of excision gaps generated by transposable elements of the 'Ac family'.

Studies on transposable elements of the Ac family have led to different models for excision gap repair in either plants or Drosophila. Excision products generated by the plant transposable elements Ac and Tam3 imply a more or less straightforward ligation of broken ends; excision products of the Drosophila P element indicate the involvement of 'double-strand break' (DSB) repair. Recent findings that excision products of Ac and Tam3 can also contain traces of the element ends indicate, however, that DSB repair might be an alternative repair mechanism in plants. A functional DSB repair mechanism in plants can also be deduced from the observed rapid increases of Ac copy number during plant development and from the involvement of Ac in the generation of internal Ac deletions. On the other hand, alternative repair mechanisms may also be functional in Drosophila, because some of the 'footprints' generated upon P excision can be explained by a mechanism that has been postulated for excision gap repair in plants. It is concluded that plants and Drosophila can use similar repair mechanisms, but that the predominance of a certain repair mechanism is determined by the host.

A functional map of the fruit-specific promoter of the tomato 2A11 gene.

The 5' region of the fruit-specific tomato gene, 2A11, contains both positive and negative regulatory elements. We divided the 5' promoter region of the 2A11 gene into small fragments, ranging in size from 211 to 634 bp and used these short DNA fragments in in vitro protein-binding studies. These studies revealed the presence of at least four fruit-specific and one leaf- and fruit-active protein-binding domains. These promoter fragments, as well as other overlapping fragments, were tested for their ability to enhance expression from a truncated heterologous promoter in transgenic plants. This analysis showed the presence of four fruit-specific and three general or leaf-active positive regulatory elements. Comparison of the results obtained with these two approaches allowed us to draw a functional map of the 2A11 promoter.

Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue.

We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted.

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Strong negative and positive regulatory elements contribute to the high-level fruit-specific expression of the tomato 2A11 gene.

Fruit-specific expression of beta-glucuronidase (GUS) activity was produced in transgenic tomato plants when the GUS-coding region was flanked by 5' and 3' regions of the tomato 2A11 gene. Deletion studies on the 5' region revealed a number of strong regulatory elements involved in the proper expression of the 2A11 gene. A 4.0 kb and a 1.3 kb 5' region can confer high-level fruit-specific GUS expression, while a 1.8 kb 5' region produces no GUS activity in leaf or fruit tissue. Thus, a strong negative regulatory element is present in the region between 1324 bp and 1796 bp upstream of the 2A11 transcriptional start and a strong fruit-specific positive regulatory element is present more than 1.8 kb upstream of the transcriptional start site. The 1.8 kb promoter region can be activated by the upstream insertion of the CaMV 35S enhancer sequence, albeit not in a fruit-specific fashion. Substitution of the 3' region of the 2A11 gene with a different 3' region does not seem to affect GUS expression significantly, indicating a minor role, if any, for the 3' region in the fruit-specific expression of the 2A11 gene.

Mutational analysis of the conserved domains of a T-region border repeat of Agrobacterium tumefaciens.

Left- and right-border repeats, which surround the T-region, contain two conserved regions separated by 5 bp that are not conserved. At the onset of T-DNA processing virD-encoded proteins introduce a nick in the largest of these conserved regions (12 bp) at a specific position in the bottom strand between a guanine and thymine nucleotide [2, 33]. In this paper we describe the effect of several site-directed mutations in the right-border repeat on tumorigenicity of Agrobacterium in plants. Our data show that mutations introduced directly around the nick site do not seriously affect the tumorigenicity of Agrobacterium, whereas mutations in the right part of this 12 bp conserved region do so. Furthermore, it appeared that the second conserved region (5 bp) is also essential for border activity and that the distance between the two conserved regions is important to obtain optimal border activity.

Bidirectional transfer from a 24 bp border repeat of Agrobacterium tumefaciens.

T-region transfer from wild-type Agrobacterium strains is thought to be an orientated process, starting at the right border repeat and terminating at the left border repeat of the T-region. Here we demonstrate that a right border repeat in the inverted orientation relative to the onc-genes can also mediate transfer of the T-region to the plant cell, although with lower efficiency as a border repeat in the native orientation. Transfer mediated by an inverted right border repeat is stimulated by the presence of the T-region transfer enhancer. Similar single stranded molecules, comprising the bottom strand of the T-DNA, were isolated from acetosyringone induced bacteria, irrespective of the orientation of the right border. These findings show that border repeats work bidirectionally to some extent.

Function of heterologous and pseudo border repeats in T region transfer via the octopine virulence system of Agrobacterium tumefaciens.

The successful transfer of the Ti plasmid T region to the plant cell is mediated by its 24 bp border repeats. Processing of the T-region prior to transfer to the plant cell is started at the right border repeat and is stimulated by a transfer enhancer sequence called "overdrive". Left and right border repeats differ somewhat in nucleotide sequence; moreover, the repeats of different Ti and Ri plasmids are slightly different. Our data indicate that these differences do not have a significant influence on border activity. However, the overdrive sequence is essential for the efficient transfer of a T region via an octopine transfer system. Our data suggest that an overdrive sequence must also be present next to the right border repeats of the nopaline Ti plasmid and the agropine of octopine and nopaline Ti plasmids express some differences in T-DNA processing activities. of cotopine and nopaline Ti plasmids express some differences in T-DNA processing activities.Furthermore, we demonstrate that certain pseudo border repeats, sequences that resemble the native 24 bp border repeat and naturally occur within the octopine Ti plasmid T-region, are able to mediate T region transfer to the plant cell, albeit with much reduced efficiency as compared to wild-type border repeats.