Changing epidemiology of methicillin-resistant Staphylococcus aureus colonization in paediatric intensive-care units

Community-associated methicillin-resistant S. aureus (CA-MRSA) accounts for a growing proportion of hospital-onset infections, and colonization is a risk factor. This study aimed to determine changes in the prevalence of CA-MRSA colonization in paediatric intensive-care units (ICUs). A total of 495 paediatric patients colonized with MRSA from neonatal, medical, surgical, and cardiac ICUs between 2001 and 2009 were identified. Isolates were characterized by spa type, staphylococcal cassette chromosome (SCC) mec type and the presence of the genes encoding Panton–Valentine leukocidin (PVL). The proportion of patients colonized with MRSA remained stable (average 3·2%). The proportion of isolates with spa type 1, SCCmec type IV and PVL increased over time to maximums in 2009 of 36·1% (P < 0·001), 54·2% (P = 0·03) and 28·9% (P = 0·003), respectively. Antibiotic susceptibility patterns showed increasing proportions susceptible to clindamycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole (P values <0·001). In conclusion, the proportion of MRSA-colonized children in ICUs with CA-MRSA increased significantly over time

Nuclear Import and Export Signals of Human Cohesins SA1/STAG1 and SA2/STAG2 Expressed in Saccharomyces cerevisiae

Abstract Background: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. Methodology/Principal Findings: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the Nterminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. Conclusions/Significance: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells

Efficient Nuclear Transport of Structurally Disturbed Cargo: Mutations in a Cargo Protein Switch Its Cognate Karyopherin

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus

RAD51 foci as a biomarker predictive of platinum chemotherapy response in ovarian cancer

PURPOSE: To determine the ability of RAD51 foci to predict platinum chemotherapy response in high-grade serous ovarian cancer (HGSOC) patient-derived samples. EXPERIMENTAL DESIGN: RAD51 and γH2AX nuclear foci were evaluated by immunofluorescence in HGSOC patient-derived cell lines (n = 5), organoids (n = 11), and formalin-fixed, paraffin-embedded tumor samples (discovery n = 31, validation n = 148). Samples were defined as RAD51-High if \u3e10% of geminin-positive cells had ≥5 RAD51 foci. Associations between RAD51 scores, platinum chemotherapy response, and survival were evaluated. RESULTS: RAD51 scores correlated with in vitro response to platinum chemotherapy in established and primary ovarian cancer cell lines (Pearson r = 0.96, P = 0.01). Organoids from platinum-nonresponsive tumors had significantly higher RAD51 scores than those from platinum-responsive tumors (P \u3c 0.001). In a discovery cohort, RAD51-Low tumors were more likely to have a pathologic complete response (RR, 5.28; P \u3c 0.001) and to be platinum-sensitive (RR, ∞; P = 0.05). The RAD51 score was predictive of chemotherapy response score [AUC, 0.90; 95% confidence interval (CI), 0.78-1.0; P \u3c 0.001). A novel automatic quantification system accurately reflected the manual assay (92%). In a validation cohort, RAD51-Low tumors were more likely to be platinum-sensitive (RR, ∞; P \u3c 0.001) than RAD51-High tumors. Moreover, RAD51-Low status predicted platinum sensitivity with 100% positive predictive value and was associated with better progression-free (HR, 0.53; 95% CI, 0.33-0.85; P \u3c 0.001) and overall survival (HR, 0.43; 95% CI, 0.25-0.75; P = 0.003) than RAD51-High status. CONCLUSIONS: RAD51 foci are a robust marker of platinum chemotherapy response and survival in ovarian cancer. The utility of RAD51 foci as a predictive biomarker for HGSOC should be tested in clinical trials

A Full Suite of Histone and Histone Modifying Genes Are Transcribed in the Dinoflagellate Lingulodinium

BACKGROUND: Dinoflagellates typically lack histones and nucleosomes are not observed in DNA spreads. However, recent studies have shown the presence of core histone mRNA sequences scattered among different dinoflagellate species. To date, the presence of all components required for manufacturing and modifying nucleosomes in a single dinoflagellate species has not been confirmed. METHODOLOGY AND RESULTS: Analysis of a Lingulodinium transcriptome obtained by Illumina sequencing of mRNA shows several different copies of each of the four core histones as well as a suite of histone modifying enzymes and histone chaperone proteins. Phylogenetic analysis shows one of each Lingulodinium histone copies belongs to the dinoflagellate clade while the second is more divergent and does not share a common ancestor. All histone mRNAs are in low abundance (roughly 25 times lower than higher plants) and transcript levels do not vary over the cell cycle. We also tested Lingulodinium extracts for histone proteins using immunoblotting and LC-MS/MS, but were unable to confirm histone expression at the protein level. CONCLUSION: We show that all core histone sequences are present in the Lingulodinium transcriptome. The conservation of these sequences, even though histone protein accumulation remains below currently detectable levels, strongly suggests dinoflagellates possess histones

Identification of a Phosphorylation-Dependent Nuclear Localization Motif in Interferon Regulatory Factor 2 Binding Protein 2

Background - Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. // Methodology/Principal Findings - Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C2C12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C2C12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. // Conclusions/Significance - Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation

Importin α7 Is Essential for Zygotic Genome Activation and Early Mouse Development

Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes

Conservation of Complex Nuclear Localization Signals Utilizing Classical and Non-Classical Nuclear Import Pathways in LANA Homologs of KSHV and RFHV

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency

Epigenetic Transcriptional Regulation of the Growth Arrest-Specific gene 1 (Gas1) in Hepatic Cell Proliferation at Mononucleosomal Resolution

BACKGROUND: Gas1 (growth arrest-specific 1) gene is known to inhibit cell proliferation in a variety of models, but its possible implication in regulating quiescence in adult tissues has not been examined to date. The knowledge of how Gas1 is regulated in quiescence may contribute to understand the deregulation occurring in neoplastic diseases. METHODOLOGY/PRINCIPAL FINDINGS: Gas1 expression has been studied in quiescent murine liver and during the naturally synchronized cell proliferation after partial hepatectomy. Chromatin immunoprecipitation at nucleosomal resolution (Nuc-ChIP) has been used to carry out the study preserving the in vivo conditions. Transcription has been assessed at real time by quantifying the presence of RNA polymerase II in coding regions (RNApol-ChIP). It has been found that Gas1 is expressed not only in quiescent liver but also at the cell cycle G(1)/S transition. The latter expression peak had not been previously reported. Two nucleosomes, flanking a nucleosome-free region, are positioned close to the transcription start site. Both nucleosomes slide in going from the active to the inactive state and vice versa. Nuc-ChIP analysis of the acquisition of histone epigenetic marks show distinctive features in both active states: H3K9ac and H3K4me2 are characteristic of transcription in G(0) and H4R3me2 in G(1)/S transition. Sequential-ChIP analysis revealed that the "repressing" mark H3K9me2 colocalize with several "activating" marks at nucleosome N-1 when Gas1 is actively transcribed suggesting a greater plasticity of epigenetic marks than proposed until now. The recruitment of chromatin-remodeling or modifying complexes also displayed distinct characteristics in quiescence and the G(1)/S transition. CONCLUSIONS/SIGNIFICANCE: The finding that Gas1 is transcribed at the G(1)/S transition suggests that the gene may exert a novel function during cell proliferation. Transcription of this gene is modulated by specific "activating" and "repressing" epigenetic marks, and by chromatin remodeling and histone modifying complexes recruitment, at specific nucleosomes in Gas1 promoter

Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex