PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.     

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis

The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage‐dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co‐repressor complex function, increased levels of c‐Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.     

UniqTag: Content-Derived Unique and Stable Identifiers for Gene Annotation

When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative k-mer, a string of length k, selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to ten builds of the Ensembl human genome spanning eight years to demonstrate this stability. The implementation of UniqTag in Ruby and an R package are available at https://github.com/sjackman/uniqtag sjackman/uniqtag. The R package is also available from CRAN: install.packages ("uniqtag"). Supplementary material and code to reproduce it is available at https://github.com/sjackman/uniqtag-paper.     

Effect of long-term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce

Long-term agricultural fertilization strategies gradually change soil properties including the associated microbial communities. Cultivated crops recruit beneficial microbes from the surrounding soil environment via root exudates. In this study, we aimed to investigate the effects of long-term fertilization strategies across field sites on the rhizosphere prokaryotic (Bacteria and Archaea) community composition and plant performance. We conducted growth chamber experiments with lettuce (Lactuca sativa L.) cultivated in soils from two long-term field experiments, each of which compared organic versus mineral fertilization strategies. 16S rRNA gene amplicon sequencing revealed the assemblage of a rhizosphere core microbiota shared in all lettuce plants across soils, going beyond differences in community composition depending on field site and fertilization strategies. The enhanced expression of several plant genes with roles in oxidative and biotic stress signalling pathways in lettuce grown in soils with organic indicates an induced physiological status in plants. Lettuce plants grown in soils with different fertilization histories were visibly free of stress symptoms and achieved comparable biomass. This suggests a positive aboveground plant response to belowground plant–microbe interactions in the rhizosphere. Besides effects of fertilization strategy and field site, our results demonstrate the crucial role of the plant in driving rhizosphere microbiota assemblage.     

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.