Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were radily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors

A systematic review of the evidence for single stage and two stage revision of infected knee replacement

BACKGROUND: Periprosthetic infection about the knee is a devastating complication that may affect between 1% and 5% of knee replacement. With over 79 000 knee replacements being implanted each year in the UK, periprosthetic infection (PJI) is set to become an important burden of disease and cost to the healthcare economy. One of the important controversies in treatment of PJI is whether a single stage revision operation is superior to a two-stage procedure. This study sought to systematically evaluate the published evidence to determine which technique had lowest reinfection rates. METHODS: A systematic review of the literature was undertaken using the MEDLINE and EMBASE databases with the aim to identify existing studies that present the outcomes of each surgical technique. Reinfection rate was the primary outcome measure. Studies of specific subsets of patients such as resistant organisms were excluded. RESULTS: 63 studies were identified that met the inclusion criteria. The majority of which (58) were reports of two-stage revision. Reinfection rated varied between 0% and 41% in two-stage studies, and 0% and 11% in single stage studies. No clinical trials were identified and the majority of studies were observational studies. CONCLUSIONS: Evidence for both one-stage and two-stage revision is largely of low quality. The evidence basis for two-stage revision is significantly larger, and further work into direct comparison between the two techniques should be undertaken as a priority

The Chemotherapeutic Drug 5-Fluorouracil Promotes PKR-Mediated Apoptosis in a p53- Independent Manner in Colon and Breast Cancer Cells

The chemotherapeutic drug 5-FU is widely used in the treatment of a range of cancers, but resistance to the drug remains a major clinical problem. Since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in 5-FU-induced apoptosis is of main clinical interest. We have identified the ds-RNA-dependent protein kinase (PKR) as a key molecular target of 5-FU involved in apoptosis induction in human colon and breast cancer cell lines. PKR distribution and activation, apoptosis induction and cytotoxic effects were analyzed during 5-FU and 5-FU/IFNα treatment in several colon and breast cancer cell lines with different p53 status. PKR protein was activated by 5-FU treatment in a p53-independent manner, inducing phosphorylation of the protein synthesis translation initiation factor eIF-2α and cell death by apoptosis. Furthermore, PKR interference promoted a decreased response to 5-FU treatment and those cells were not affected by the synergistic antitumor activity of 5-FU/IFNα combination. These results, taken together, provide evidence that PKR is a key molecular target of 5-FU with potential relevance in the clinical use of this drug

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces

Identifying Determinants of Cullin Binding Specificity Among the Three Functionally Different Drosophila melanogaster Roc Proteins via Domain Swapping

BACKGROUND: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known. METHODOLOGY/PRINCIPAL FINDINGS: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well. CONCLUSIONS: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo

Differential baseline and response profile to IFN-γ gene transduction of IL-6/IL-6 receptor-α secretion discriminate primary tumors versus bone marrow metastases of nasopharyngeal carcinomas in culture

<p>Abstract</p> <p>Background</p> <p>Understanding of immunobiology of bone marrow metastases (designated BM-NPC) <it>versus </it>primary tumors (P-NPC) of the nasopharynx is far from complete. The aim of this study was to determine if there would be differences between cultured P-NPCs and BM-NPCs with respect to (i) constitutive IL-6 and the IL-6 receptor gp80 subunit (IL-6Rα) levels in the spent media of nontransduced cells, and (ii) IL-6 and IL-6Rα levels in the spent media of cells transduced with a retroviral vector containing the <it>IFN-γ </it>gene.</p> <p>Methods</p> <p>A panel of NPC cell lines were transduced with the <it>IFN-γ </it>gene through a retroviral vector. Four clonal sublines were isolated <it>via </it>limiting dilution methods. Cytofluorometric analysis was performed for the detection of cell surface antigens of HLA class I, HLA class II and ICAM-1. ELISA was used to assay for IFN-γ, IL-6 and IL-6Rα in the spent media of cultured cell lines.</p> <p>Results</p> <p>Our results showed that in day 3 culture supernatants, low levels of soluble IL-6 were detected in 5/5 cultured tumors derived from P-NPCs, while much higher constitutive levels of IL-6 were detected in 3/3 metastasis-derived NPC cell lines including one originated from ascites; the difference was significant (<it>p </it>= 0.025). An inverse relationship was found between IL-6Rα and IL-6 in their release levels in cultured P-NPCs and metastasis-derived NPCs. In <it>IFN-γ</it>-transduced-P-NPCs, IL-6 production increased and yet IL-6Rα decreased substantially, as compared to nontransduced counterparts. At variance with P-NPC cells, the respective ongoing IL-6 and IL-6Rα release patterns of BM-NPC cells were not impeded as much following <it>IFN-γ </it>transduction. These observations were confirmed by extended kinetic studies with representative NPC cell lines and clonal sublines. The latter observation with the clonal sublines also indicates that selection for high IL-6 or low IL-6Rα producing subpopulations did not occur as a result of <it>IFN-γ</it>-transduction process. P-NPCs, which secreted constitutively only marginal levels of IFN-γ (8.4 ~ 10.5 pg/ml), could be enhanced to produce higher levels of IFN-γ (6.8- to 10.3-fold increase) after <it>IFN-γ </it>transduction. Unlike P-NPCs, BM-NPCs spontaneously released IFN-γ at moderate levels (83.8 ~ 100.7 pg/ml), which were enhanced by 1.3- to 2.2-fold in the spent media of their <it>IFN-γ</it>-transduced counterparts.</p> <p>Conclusion</p> <p>Our results showed that cultured P-NPCs and BM-NPCs could be distinguished from one another on the basis of their differential baseline secretion pattern of IFN-γ, IL-6 and IL-6Rα, and their differential response profiles to <it>IFN-γ </it>gene transfer of the production of these three soluble molecules. These results suggest that the IL-6 and IFN-γ pathways in a background of genetic instability be involved in the acquisition of metastatic behaviour in BM-NPCs.</p

Outcome of prosthesis exchange for infected knee arthroplasty: the effect of treatment approach: A systematic review of the literature

Background and purpose Two-stage revision remains the gold standard in the treatment of infected knee arthroplasty. Lately, good long-term results of direct exchange arthroplasty have been reported. The purpose of this literature review is to compare the clinical outcome achieved with one-stage revision and two-stage revision with different types of spacers

Selective Survival and Maturation of Adult-Born Dentate Granule Cells Expressing the Immediate Early Gene Arc/Arg3.1

Progenitor cells in the adult dentate gyrus provide a constant supply of neuronal precursors, yet only a small fraction of these cells survive and develop into mature dentate granule cells (DGCs). A major challenge of current research is thus to understand the stringent selection process that governs the maturation and functional integration of adult-born DGCs. In mature DGCs, high-frequency stimulation (HFS) of the perforant path input elicits robust expression of the immediate early gene Arc/Arg3.1, trafficking of its mRNA to dendrites, and local synthesis of the protein necessary for consolidation of long-term potentiation (LTP). Given the synaptic commitment inherent in LTP consolidation, we considered that HFS-evoked expression of Arc could be used to timemap the functional integration of newborn DGCs. Dividing cells were birthmarked by BrdU-labeling at 1, 7, 14, 21, or 28 days prior to induction of LTP and expression of Arc was examined by confocal microscopy. Contrary to expectation, LTP did not induce Arc expression in newborn cells at any age, suggesting they might be refractory to synaptically-evoked Arc expression for at least one month. Importantly, however, spontaneous expression of Arc was detected in BrdU-labeled cells and strongly associated with the survival and maturation of NeuN-positive DGCs. Moreover, Arc expression at the earliest ages (1 and 7 days), clearly precedes the formation of glutamatergic synapses on new neurons. These results suggest an unexpected early role for Arc in adult-born DGCs, distinct from its functions in LTP, LTD, and homeostatic synaptic plasticity

Impairment of circulating endothelial progenitors in Down syndrome

<p>Abstract</p> <p>Background</p> <p>Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.</p> <p>Methods</p> <p>Circulating endothelial progenitors of Down syndrome affected individuals were isolated, <it>in vitro </it>cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of <it>CXCL12 </it>gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.</p> <p>Results</p> <p>We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.</p> <p>Conclusions</p> <p>Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.</p

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion