Regulation of S-layer synthesis and secretion in Caulobacter crescentus

Caulobacter crescentus is a non-pathogenic, Gram negative bacterium that forms a surface layer (S-layer) that is composed exclusively of the protein RsaA in a crystalline array. RsaA, which constitutes 10-12% of total cellular protein, is secreted by a robust type I protein transport system in C. crescentus. These features have made this bacterium an attractive candidate for biotechnology applications that require the production of proteins of interest at high levels and purity. In a previous study aimed at determining the maximal protein secretion capability of the type I system in C. crescentus, the overexpression of rsaA did not result in the increased production and secretion of RsaA. However, upon further investigation it was determined that the plasmid used to overexpress rsaA included an extended 5' untranslated region (UTR). The results presented herein suggest that this extended 5' UTR caused a decrease in the half-life of rsaA mRNA to ~19 minutes compared to the ~36 minutes half-life of wild type rsaA mRNA which may in turn explain the lack of increased RsaA production. By contrast, production and secretion of RsaA was significantly increased (2.2 ± 0.1 fold) in C. crescentus transformed with a plasmid containing rsaA without an extended 5' UTR when compared to wild type. Deletion of the outer membrane transporters, RsaFa and RsaFb, prevented the secretion of RsaA and resulted in a significant down-regulation of RsaA production. By using quantitative reverse-transcriptase PCR (qRT-PCR) it was determined that the amount of rsaA mRNA in the transporter deletion mutant was similar to wild type (0.9 ± 0.1-fold of wild type). This suggests that the down-regulation of RsaA observed in these mutants occurred at a posttranscriptional level. Previous experiments showed that recombinant forms of RsaA containing an abundance of positively charged amino acids were not detected at the cell surface, indicating a complete inhibition of secretion. Three dimensional models of RsaFa and RsaFb using the Swiss Model program placed twelve negatively charged amino acids near the entrance of the predicted pore structure on the periplasmic side. In order to test the hypothesis that these negatively charged amino acid residues were inhibiting the secretion of recombinant RsaA, site-directed mutagenesis was used to alter them. However, none of the mutants relieved the inhibition of recombinant RsaA secretion. Moreover, three of the mutants, RsaFb-D395A, RsaFb-E185A/D395A, and RsaFb-D395A/E402A, also inhibited the secretion of wild type RsaA. Taken together, these results demonstrate that RsaA expression can be upregulated and that the type 1 secretion system of C. crescentus can facilitate this increase. In addition, regulation of RsaA can occur at a posttranscriptional level when its secretion is blocked, as is the case in the outer membrane transporter deletion mutants. Furthermore, site-directed mutagenesis suggests a role for negatively charged amino acids in the secretion of S-layer protein in RsaFb but not RsaFa.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Gender differences in clusters of NPS in Dutch nursing homes - a factor analysis

Item does not contain fulltextBACKGROUND: Neuropsychiatric symptoms (NPS) have a high prevalence among patients with dementia, up to 80%. NPS can be grouped by type and stage of dementia. However, NPS have not previously been grouped by gender. Our objective was to investigate whether NPS cluster differently in men or women in the nursing home patients. METHODS: Factor analysis to assess the clustering of items in the Cohen-Mansfield Agitation Inventory (CMAI) and Neuropsychiatric Inventory-Nursing home version (NPI-NH) into components, for both scales and for gender. Differences in symptom clustering between male and female patients were assessed using a three-step procedure: (1) identifying a gender specific distinctive item, (2) describe the correlation between the distinctive item with any other item in this cluster, (3) testing whether the correlation between a distinctive item and any other item in the cluster (which is present in both sexes) is different for males and females using a general linear model. RESULTS: Our database consisted of 1,609 patients. There were five male and three female clusters for NPI-NH and eight male and seven female clusters for CMAI. There were three distinctive items in the NPI-NH and ten in the CMAI. CONCLUSIONS: There are other clusters of NPS in males and females. Our analysis revealed more significant relations in female than male patients. This might have an implication on the clinical course

Differential coreceptor expression allows for independent evolution of non–syncytium-inducing and syncytium-inducing HIV-1

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(–) and CCR5(–) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways

Validation of a physician global assessment tool for vitiligo extent: Results of an international vitiligo expert meeting

Currently, vitiligo lacks a validated Physician Global Assessment (PGA) for disease extent. This PGA can be used to stratify and interpret the numeric scores obtained by the Vitiligo Extent Score (VES). We investigated the interrater reliability of a 5-point PGA scale during an international vitiligo workshop. Vitiligo experts from five different continents rated photographs of non-segmental vitiligo patients with varying degrees of extent with the PGA score. Good interrater agreements (intraclass correlation coefficient >0.6) were observed between the raters overall and within each continent. All hypotheses to evaluate construct validity were confirmed. Median VES values per category were for limited 1.10 [IQR: 0.21–1.67], moderate 3.17 [IQR: 1.75–6.21], extensive 9.58 [IQR: 6.21–13.03] and very extensive 42.67 [IQR: 21.20–42.67]. Defined categories for vitiligo extent can be valuable for inclusion criteria and may impact future reimbursement criteria