Visualizing the Needle in the Haystack: In Situ Hybridization With Fluorescent Dendrimers

In situ hybridization with 3DNA™ dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues

Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1) activity and increase airway smooth muscle contraction in asthma

Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms

Variation in mitochondrial function in hypoxia-sensitive and hypoxia-tolerant human glioma cells

We have shown previously that human glioblastoma multiforme cells vary in their ability to survive under hypoxic conditions. Under oxygen limiting conditions, hypoxia-tolerant cells decrease their oxygen consumption rate whereas hypoxia-sensitive cells continue to consume oxygen at a relatively steady rate until the oxygen supply becomes exhausted. We now show that hypoxia-tolerant and hypoxia-sensitive cells exhibit distinct patterns of mitochondrial function in response to hypoxic challenge. Hypoxia-tolerant cell lines retain stable mitochondrial membrane potential and ATP concentration when incubated under oxygen limiting conditions. In addition, hypoxia-tolerant cell lines are consistently more sensitive to a wide spectrum of inhibitors of mitochondrial function than are hypoxia-sensitive cells. In contrast, the hypoxia-sensitive cells are unable to maintain stable mitochondrial membrane potential and ATP levels when incubated at reduced oxygen tension. These results demonstrate significant differences in the mitochondrial function between these two phenotypes and reinforce previous data that suggest a regulatory role for mitochondria in the development of hypoxia tolerance

Chemotherapeutic errors in hospitalised cancer patients: attributable damage and extra costs

<p>Abstract</p> <p>Background</p> <p>In spite of increasing efforts to enhance patient safety, medication errors in hospitalised patients are still relatively common, but with potentially severe consequences. This study aimed to assess antineoplastic medication errors in both affected patients and intercepted cases in terms of frequency, severity for patients, and costs.</p> <p>Methods</p> <p>A 1-year prospective study was conducted in order to identify the medication errors that occurred during chemotherapy treatment of cancer patients at a French university hospital. The severity and potential consequences of intercepted errors were independently assessed by two physicians. A cost analysis was performed using a simulation of potential hospital stays, with estimations based on the costs of diagnosis-related groups.</p> <p>Results</p> <p>Among the 6, 607 antineoplastic prescriptions, 341 (5.2%) contained at least one error, corresponding to a total of 449 medication errors. However, most errors (n = 436) were intercepted before medication was administered to the patients. Prescription errors represented 91% of errors, followed by pharmaceutical (8%) and administration errors (1%). According to an independent estimation, 13.4% of avoided errors would have resulted in temporary injury and 2.6% in permanent damage, while 2.6% would have compromised the vital prognosis of the patient, with four to eight deaths thus being avoided. Overall, 13 medication errors reached the patient without causing damage, although two patients required enhanced monitoring. If the intercepted errors had not been discovered, they would have resulted in 216 additional days of hospitalisation and cost an estimated annual total of 92, 907€, comprising 69, 248€ (74%) in hospital stays and 23, 658€ (26%) in additional drugs.</p> <p>Conclusion</p> <p>Our findings point to the very small number of chemotherapy errors that actually reach patients, although problems in the chemotherapy ordering process are frequent, with the potential for being dangerous and costly.</p

Application of isothermal titration calorimetry in evaluation of protein–nanoparticle interactions

Nanoparticles (NPs) offer a number of advantages over small organic molecules for controlling protein behaviour inside the cell. Protein binding to the surface of NPs depends on their surface characteristics, composition and method of preparation (Mandal et al. in J Hazard Mater 248–249:238–245, 2013). It is important to understand the binding affinities, stoichiometries and thermodynamical parameters of NP–protein interactions in order to see which interaction will have toxic and hazardous consequences and thus to prevent it. On the other side, because proteins are on the brink of stability, they may experience interactions with some types of NPs that are strong enough to cause denaturation or significantly change their conformations with concomitant loss of their biological function. Structural changes in the protein may cause exposure of new antigenic sites, “cryptic” peptide epitopes, potentially triggering an immune response which can promote autoimmune disease (Treuel et al. in ACS Nano 8(1):503–513, 2014). Mechanistic details of protein structural changes at NP surface have still remained elusive. Understanding the formation and persistence of the protein corona is critical issue; however, there are no many analytical methods which could provide detailed information about the NP–protein interaction characteristics and about protein structural changes caused by interactions with nanoparticles. The article reviews recent studies in NP–protein interactions research and application of isothermal titration calorimetry (ITC) in this research. The study of protein structural changes upon adsorption on nanoparticle surface and application of ITC in these studies is emphasized. The data illustrate that ITC is a versatile tool for evaluation of interactions between NPs and proteins. When coupled with other analytical methods, it is important analytical tool for monitoring conformational changes in proteins

Theoretical analysis of the dose dependence of the oxygen enhancement ratio and its relevance for clinical applications

<p>Abstract</p> <p>Background</p> <p>The increased resistance of hypoxic cells to ionizing radiation is usually believed to be the primary reason for treatment failure in tumors with oxygen-deficient areas. This oxygen effect can be expressed quantitatively by the oxygen enhancement ratio (OER). Here we investigate theoretically the dependence of the OER on the applied local dose for different types of ionizing irradiation and discuss its importance for clinical applications in radiotherapy for two scenarios: small dose variations during hypoxia-based dose painting and larger dose changes introduced by altered fractionation schemes.</p> <p>Methods</p> <p>Using the widespread Alper-Howard-Flanders and standard linear-quadratic (LQ) models, OER calculations are performed for T1 human kidney and V79 Chinese hamster cells for various dose levels and various hypoxic oxygen partial pressures (pO2) between 0.01 and 20 mmHg as present in clinical situations <it>in vivo</it>. Our work comprises the analysis for both low linear energy transfer (LET) treatment with photons or protons and high-LET treatment with heavy ions. A detailed analysis of experimental data from the literature with respect to the dose dependence of the oxygen effect is performed, revealing controversial opinions whether the OER increases, decreases or stays constant with dose.</p> <p>Results</p> <p>The behavior of the OER with dose per fraction depends primarily on the ratios of the LQ parameters alpha and beta under hypoxic and aerobic conditions, which themselves depend on LET, pO2 and the cell or tissue type. According to our calculations, the OER variations with dose <it>in vivo </it>for low-LET treatments are moderate, with changes in the OER up to 11% for dose painting (1 or 3 Gy per fraction compared to 2 Gy) and up to 22% in hyper-/hypofractionation (0.5 or 20 Gy per fraction compared to 2 Gy) for oxygen tensions between 0.2 and 20 mmHg typically measured clinically in hypoxic tumors. For extremely hypoxic cells (0.01 mmHg), the dose dependence of the OER becomes more pronounced (up to 36%). For high LET, OER variations up to 4% for the whole range of oxygen tensions between 0.01 and 20 mmHg were found, which were much smaller than for low LET.</p> <p>Conclusions</p> <p>The formalism presented in this paper can be used for various tissue and radiation types to estimate OER variations with dose and help to decide in clinical practice whether some dose changes in dose painting or in fractionation can bring more benefit in terms of the OER in the treatment of a specific hypoxic tumor.</p

Characterization of the past and current duplication activities in the human 22q11.2 region

<p>Abstract</p> <p>Background</p> <p>Segmental duplications (SDs) on 22q11.2 (LCR22), serve as substrates for meiotic non-allelic homologous recombination (NAHR) events resulting in several clinically significant genomic disorders.</p> <p>Results</p> <p>To understand the duplication activity leading to the complicated SD structure of this region, we have applied the A-Bruijn graph algorithm to decompose the 22q11.2 SDs to 523 fundamental duplication sequences, termed subunits. Cross-species syntenic analysis of primate genomes demonstrates that many of these LCR22 subunits emerged very recently, especially those implicated in human genomic disorders. Some subunits have expanded more actively than others, and young <it>Alu </it>SINEs, are associated much more frequently with duplicated sequences that have undergone active expansion, confirming their role in mediating recombination events. Many copy number variations (CNVs) exist on 22q11.2, some flanked by SDs. Interestingly, two chromosome breakpoints for 13 CNVs (mean length 65 kb) are located in paralogous subunits, providing direct evidence that SD subunits could contribute to CNV formation. Sequence analysis of PACs or BACs identified extra CNVs, specifically, 10 insertions and 18 deletions within 22q11.2; four were more than 10 kb in size and most contained young <it>AluY</it>s at their breakpoints.</p> <p>Conclusions</p> <p>Our study indicates that <it>AluY</it>s are implicated in the past and current duplication events, and moreover suggests that DNA rearrangements in 22q11.2 genomic disorders perhaps do not occur randomly but involve both actively expanded duplication subunits and <it>Alu </it>elements.</p

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation