Application of GFAT as a Novel Selection Marker to Mediate Gene Expression

The enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT), also known as glucosamine synthase (GlmS), catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. For the first time, the GFAT gene was proven to possess a function as an effective selection marker for genetically modified (GM) microorganisms. This was shown by construction and analysis of two GFAT deficient strains, E. coli ΔglmS and S. pombe Δgfa1, and the ability of the GFAT encoding gene to mediate plasmid selection. The gfa1 gene of the fission yeast Schizosaccharomyces pombe was deleted by KanMX6-mediated gene disruption and the Cre-loxP marker removal system, and the glmS gene of Escherichia coli was deleted by using λ-Red mediated recombinase system. Both E. coli ΔglmS and S. pombe Δgfa1 could not grow normally in the media without addition of glucosamine. However, the deficiency was complemented by transforming the plasmids that expressed GFAT genes. The xylanase encoding gene, xynA2 from Thermomyces lanuginosus was successfully expressed and secreted by using GFAT as selection marker in S. pombe. Optimal glucosamine concentration for E. coli ΔglmS and S. pombe Δgfa1 growth was determined respectively. These findings provide an effective technique for the construction of GM bacteria without an antibiotic resistant marker, and the construction of GM yeasts to be applied to complex media

The World Starts With Me: using intervention mapping for the systematic adaptation and transfer of school-based sexuality education from Uganda to Indonesia

Evidence-based health promotion programmes, including HIV/AIDS prevention and sexuality education programmes, are often transferred to other cultures, priority groups and implementation settings. Challenges in this process include the identification of retaining core elements that relate to the programme’s effectiveness while making changes that enhances acceptance in the new context and for the new priority group. This paper describes the use of a systematic approach to programme adaptation using a case study as an example. Intervention Mapping, a protocol for the development of evidence-based behaviour change interventions, was used to adapt the comprehensive school-based sexuality education programme ‘The World Starts With Me’. The programme was developed for a priority population in Uganda and adapted to a programme for Indonesian secondary school students. The approach helped to systematically address the complexity and challenges of programme adaptation and to find a balance between preservation of essential programme elements (i.e. logic models) that may be crucial to the programme’s effectiveness, including key objectives and theoretical behaviour change methods, and the adaptation of the programme to be acceptable to the new priority group and the programme implementers

Evaluating teaching effectiveness in nursing education:An Iranian perspective

BACKGROUND: The main objective of this study was to determine the perceptions of Iranian nurse educators and students regarding the evaluation of teaching effectiveness in university-based programs. METHODS: An exploratory descriptive design was employed. 143 nurse educators in nursing faculties from the three universities in Tehran, 40 undergraduate, and 30 graduate students from Tehran University composed the study sample. In addition, deans from the three nursing faculties were interviewed. A researcher-developed questionnaire was used to determine the perceptions of both faculty and students about evaluating the teaching effectiveness of nurse educators, and an interview guide was employed to elicit the views of deans of faculties of nursing regarding evaluation policies and procedures. Data were analyzed using parametric and nonparametric statistics to identify similarities and differences in perceptions within the Iranian nurse educator group and the student group, and between these two groups of respondents. RESULTS: While faculty evaluation has always been a major part of university based nursing programs, faculty evaluation must be approached more analytically, objectively, and comprehensively to ensure that all nursing educators receive the fairest treatment possible and that the teaching-learning process is enhanced. CONCLUSION: Educators and students stressed that systematic and continuous evaluation as well as staff development should be the primary goals for the faculty evaluation process. The ultimate goals is the improvement of teaching by nurse educators

Role of Symbiotic Auxotrophy in the Rhizobium-Legume Symbioses

Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes

In vivo biofunctional evaluation of hydrogels for disc regeneration

Purpose Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. Methods In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. Results Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. Conclusions None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioningThis work was supported by the EU-project Disc Regeneration (NMP3-LA-2008-213904). Technical assistance of Iris Baum and the whole animal surgery team of the Institute of Orthopaedic Research and Biomechanics, Ulm, are gratefully acknowledged. DDAHA hydrogels were kindly provided by Cristina Longinotti (DDAHA, Anika Therapeutics, Abano Therme, Italy)

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

The role of LINEs and CpG islands in dosage compensation on the chicken Z chromosome

Most avian Z genes are expressed more highly in ZZ males than ZW females, suggesting that chromosome-wide mechanisms of dosage compensation have not evolved. Nevertheless, a small percentage of Z genes are expressed at similar levels in males and females, an indication that a yet unidentified mechanism compensates for the sex difference in copy number. Primary DNA sequences are thought to have a role in determining chromosome gene inactivation status on the mammalian X chromosome. However, it is currently unknown whether primary DNA sequences also mediate chicken Z gene compensation status. Using a combination of chicken DNA sequences and Z gene compensation profiles of 310 genes, we explored the relationship between Z gene compensation status and primary DNA sequence features. Statistical analysis of different Z chromosomal features revealed that long interspersed nuclear elements (LINEs) and CpG islands are enriched on the Z chromosome compared with 329 other DNA features. Linear support vector machine (SVM) classifiers, using primary DNA sequences, correctly predict the Z compensation status for >60% of all Z-linked genes. CpG islands appear to be the most accurate classifier and alone can correctly predict compensation of 63% of Z genes. We also show that LINE CR1 elements are enriched 2.7-fold on the chicken Z chromosome compared with autosomes and that chicken chromosomal length is highly correlated with percentage LINE content. However, the position of LINE elements is not significantly associated with dosage compensation status of Z genes. We also find a trend for a higher proportion of CpG islands in the region of the Z chromosome with the fewest dosage-compensated genes compared with the region containing the greatest concentration of compensated genes. Comparison between chicken and platypus genomes shows that LINE elements are not enriched on sex chromosomes in platypus, indicating that LINE accumulation is not a feature of all sex chromosomes. Our results suggest that CpG islands are not randomly distributed on the Z chromosome and may influence Z gene dosage compensation status

Relative impact of key sources of systematic noise in Affymetrix and Illumina gene-expression microarray experiments

<p>Abstract</p> <p>Background</p> <p>Systematic processing noise, which includes batch effects, is very common in microarray experiments but is often ignored despite its potential to confound or compromise experimental results. Compromised results are most likely when re-analysing or integrating datasets from public repositories due to the different conditions under which each dataset is generated. To better understand the relative noise-contributions of various factors in experimental-design, we assessed several Illumina and Affymetrix datasets for technical variation between replicate hybridisations of Universal Human Reference (UHRR) and individual or pooled breast-tumour RNA.</p> <p>Results</p> <p>A varying degree of systematic noise was observed in each of the datasets, however in all cases the relative amount of variation between standard control RNA replicates was found to be greatest at earlier points in the sample-preparation workflow. For example, 40.6% of the total variation in reported expressions were attributed to replicate extractions, compared to 13.9% due to amplification/labelling and 10.8% between replicate hybridisations. Deliberate probe-wise batch-correction methods were effective in reducing the magnitude of this variation, although the level of improvement was dependent on the sources of noise included in the model. Systematic noise introduced at the chip, run, and experiment levels of a combined Illumina dataset were found to be highly dependant upon the experimental design. Both UHRR and pools of RNA, which were derived from the samples of interest, modelled technical variation well although the pools were significantly better correlated (4% average improvement) and better emulated the effects of systematic noise, over all probes, than the UHRRs. The effect of this noise was not uniform over all probes, with low GC-content probes found to be more vulnerable to batch variation than probes with a higher GC-content.</p> <p>Conclusions</p> <p>The magnitude of systematic processing noise in a microarray experiment is variable across probes and experiments, however it is generally the case that procedures earlier in the sample-preparation workflow are liable to introduce the most noise. Careful experimental design is important to protect against noise, detailed meta-data should always be provided, and diagnostic procedures should be routinely performed prior to downstream analyses for the detection of bias in microarray studies.</p

Human Immunodeficiency Virus-1 Uses the Mannose-6-Phosphate Receptor to Cross the Blood-Brain Barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB