ATM mediated phosphorylation of CHD4 contributes to genome maintenance

Background: In order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling. Results: Here we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage. Conclusion: These results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.

Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.

Bioactive SrO–SiO2 glass with well-ordered mesopores : characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO2 glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO2 in an effort to develop a bioactive mesoporous SrO–SiO2 (Sr–Si) glass with the capacity to deliver Sr2+ ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr2+ on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr–Si glass were investigated. The prepared mesoporous Sr–Si glass was found to have an excellent release profile of bioactive Sr2+ ions and dexamethasone, and the incorporation of Sr2+ improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr–Si glass had no cytotoxic effects and its release of Sr2+ and SiO44− ions enhanced alkaline phosphatase activity – a marker of osteogenic cell differentiation – in human bone mesenchymal stem cells. Mesoporous Sr–Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr2+ into mesoporous SiO2 glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications. Keywords: Mesoporous Sr–Si glass; Drug delivery; Bioactivity; Bone repair; Scaffolds

The spermostatic and microbicidal actions of quinones and malemides : towards a dual-purpose contraceptive agent

There is an urgent need to develop safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases. Here we report the identification of a group of compounds that on contact with human spermatozoa induce a state of “spermostasis,” characterized by the extremely rapid inhibition of sperm movement without compromising cell viability. These spermostatic agents were more active and significantly less toxic than the reagent in current clinical use, nonoxynol 9, giving therapeutic indices (ratio of spermostatic to cytotoxic activity) that were orders of magnitude greater than this traditional spermicide. Although certain compounds could trigger reactive oxygen species generation by spermatozoa, this activity was not correlated with spermostasis. Rather, the latter was associated with alkylation of two major sperm tail proteins that were identified as A Kinase-Anchoring Proteins (AKAP3 and AKAP4) by mass spectrometry. As a consequence of disrupted AKAP function, the abilities of cAMP to drive protein kinase A-dependent activities in the sperm tail, such as the activation of SRC and the consequent stimulation of tyrosine phosphorylation, were suppressed. Furthermore, analysis of microbicidal activity using Chlamydia muridarum revealed powerful inhibitory effects at the same low micromolar doses that suppressed sperm movement. In this case, the microbicidal action was associated with alkylation of Major Outer Membrane Protein (MOMP), a major chlamydial membrane protein. Taken together, these results have identified for the first time a novel set of cellular targets and chemical principles capable of providing simultaneous defense against both fertility and the spread of sexually transmitted disease.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

SNAREing immunity : the role of SNAREs in the immune system

The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses.

An improved model of tumour-immune system interactions

The immune system plays an important role in defending the body against tumours and other threats. Currently, mechanisms involved in immune system interactions with tumour cells are not fully understood. Here we develop a mathematical tool that can be used in aiding to address this shortfall in understanding. This paper de- scribes a hybrid cellular automata model of the interaction between a growing tumour and cells of the innate and specific immune system including the effects of chemokines that builds on previous models of tumour-immune system interactions. In particular, the model is focused on the response of immune cells to tumour cells and how the dynamics of the tumour cells change due to the immune system of the host. We present results and predictions of in silico experiments including simulations of Kaplan-Meier survival-like curves.

The open window of susceptibility to infection after acute exercise in healthy young male elite athletes

The 'open window' theory is characterised by short term suppression of the immune system following an acute bout of endurance exercise. This window of opportunity may allow for an increase in susceptibility to upper respiratory illness (URI). Many studies have indicated a decrease in immune function in response to exercise. However, many studies do not indicate changes in immune function past 2 hours after the completion of exercise, consequently failing to determine whether these immune cells numbers, or importantly their function, return to resting levels before the start of another bout of exercise. Ten male 'A' grade cyclists (age 24.2 +/- 5.3 years; body mass 73.8 +/- 6.5 kg; VO(2peak) 65.9 +/- 7.1 mL.kg(-1).min(-1)) exercised for two hours at 90% of their second ventilatory threshold. Blood samples were collected pre-, immediately post-, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours post-exercise. Immune variables examined included total leukocyte counts, neutrophil function (oxidative burst and phagocytic function), lymphocyte subset counts (CD4(+), CD8(+), and CD16(+)/56(+)), natural killer cell activity (NKCA), and NK phenotypes (CD56(dim)CD16(+), and CD56(bright)CD16(-)). There was a significant increase in total lymphocyte numbers from pre-, to immediately post-exercise (p<0.01), followed by a significant decrease at 2 hours post-exercise (p<0.001). CD4(+) T-cell counts significantly increased from pre-exercise, to 4 hours post- (p<0.05), and 6 hours post-exercise (p<0.01). However, NK (CD16(+)/56(+)) cell numbers decreased significantly from pre-exercise to 4 h post-exercise (p<0.05), to 6 h post-exercise (p<0.05), and to 8 h post-exercise (p<0.01). In contrast, CD56(bright)CD16- NK cell counts significantly increased from pre-exercise to immediately post-exercise (p<0.01). Neutrophil oxidative burst activity did not significantly change in response to exercise, while neutrophil cell counts significantly increased from pre-exercise, to immediately post-exercise (p<0.05), and 2 hours post-exercise (p<0.01), and remained significantly above pre-exercise levels to 8 hours post-exercise (p<0.01). Neutrophil phagocytic function significantly decreased from 2 hours post-exercise, to 6 hours post- (p<0.05), and 24 hours post-exercise (p<0.05). Finally, eosinophil cell counts significantly increased from 2 hours post to 6 hours post- (p<0.05), and 8 hours post-exercise (p<0.05). This is the first study to show changes in immunological variables up to 8 hours post-exercise, including significant NK cell suppression, NK cell phenotype changes, a significant increase in total lymphocyte counts, and a significant increase in eosinophil cell counts all at 8 hours post-exercise. Suppression of total lymphocyte counts, NK cell counts and neutrophil phagocytic function following exercise may be important in the increased rate of URI in response to regular intense endurance training.

Drugs and the gastrointestinal tract

16.1. Agents to control acidity 16.1.1 Antacids 16.1.2 Proton pump inhibitors and antibiotics for Helicobacter pylori 16.1.3 Histamine H2 receptor antagonists 16.1.4 Misoprostol 16.1.5 Sucralfate 16.2. Prokinetics and emetics 16.2.1 Introduction to prokinetics 16.2.2 Prokinetic agents 16.2.3 Emesis with cytotoxic drugs and drugs for 16.2.4 Motion sickness and drugs for 16.2.5 Drugs for post-operative emesis 16.3. Agents used for diarrhea, constipation, irritable bowel syndrome 16.3.1 Treatment for diarrhea 16.3.2 Treatment for constipation 16.3.3 Treatment for opioid-induced constipation 16.4. Drugs for inflammatory bowel disease 16.4.1 Mesalazine 16.4.2 Glucocorticoids 16.4.3 Infliximab

An introduction to anticancer drugs

An introduction to anticancer drugs 24.1 Introduction 24.2 The rationale behind anticancer drug therapy 24.3 Drugs used in cancer 24.3.1 Alkylating agents 24.3.2 Cytotoxic antibiotics 24.3.3 Antimetabolites 24.3.4 Microtubule inhibitors 24.3.5 Monoclonal antibodies 24.3.6 Steroid hormones and their antagonists 24.3.7 Other treatments

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Gene transfer vectors targeted to human prostate cancer : do we need better preclinical testing systems?

Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.

Lynch syndrome-associated breast cancers: clinicopathologic characteristics of a case series from the colon cancer family registry

Purpose: The recognition of breast cancer as a spectrum tumor in Lynch syndrome remains controversial. The aim of this study was to explore features of breast cancers arising in Lynch syndrome families. Experimental Design: This observational study involved 107 cases of breast cancer identified from the Colorectal Cancer Family Registry (Colon CFR) from 90 families in which (a) both breast and colon cancer co-occurred, (b) families met either modified Amsterdam criteria, or had at least one early-onset (<50 years) colorectal cancer, and (c) breast tissue was available within the biospecimen repository for mismatch repair (MMR) testing. Eligibility criteria for enrollment in the Colon CFR are available online. Breast cancers were reviewed by one pathologist. Tumor sections were stained for MLH1, PMS2, MSH2, and MSH6, and underwent microsatellite instability testing. Results: Breast cancer arose in 35 mutation carriers, and of these, 18 (51%) showed immunohistochemical absence of MMR protein corresponding to the MMR gene mutation segregating the family. MMR-deficient breast cancers were more likely to be poorly differentiated (P = 0.005) with a high mitotic index (P = 0.002), steroid hormone receptor–negative (estrogen receptor, P = 0.031; progesterone receptor, P = 0.022), and to have peritumoral lymphocytes (P = 0.015), confluent necrosis (P = 0.002), and growth in solid sheets (P < 0.001) similar to their colorectal counterparts. No difference in age of onset was noted between the MMR-deficient and MMR-intact groups. Conclusions: MMR deficiency was identified in 51% of breast cancers arising in known mutation carriers. Breast cancer therefore may represent a valid tissue option for the detection of MMR deficiency in which spectrum tumors are lacking