Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3.

Interleukin-1 receptor-associated kinase-3 (IRAK3) is a critical checkpoint molecule of inflammatory responses in the innate immune system. The pseudokinase domain of IRAK3 contains a guanylate cyclase (GC) centre that generates small amounts of cyclic guanosine monophosphate (cGMP) associated with IRAK3 functions in inflammation. However, the mechanisms of IRAK3 actions are poorly understood. The effects of low cGMP levels on inflammation are unknown, therefore a dose-response effect of cGMP on inflammatory markers was assessed in THP-1 monocytes challenged with lipopolysaccharide (LPS). Sub-nanomolar concentrations of membrane permeable 8-Br-cGMP reduced LPS-induced NFκB activity, IL-6 and TNF-α cytokine levels. Pharmacologically upregulating cellular cGMP levels using a nitric oxide donor reduced cytokine secretion. Downregulating cellular cGMP using a soluble GC inhibitor increased cytokine levels. Knocking down IRAK3 in THP-1 cells revealed that unlike the wild type cells, 8-Br-cGMP did not suppress inflammatory responses. Complementation of IRAK3 knockdown cells with wild type IRAK3 suppressed cytokine production while complementation with an IRAK3 mutant at GC centre only partially restored this function. Together these findings indicate low levels of cGMP form a critical component in suppressing cytokine production and in mediating IRAK3 action, and this may be via a cGMP enriched nanodomain formed by IRAK3 itself.

Intrinsic and extrinsic apoptosis responses in leukaemia cells following daunorubicin treatment.

BACKGROUND: Daunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course. METHODS: This study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins. RESULTS: Daunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells. CONCLUSIONS: This study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia.

BACKGROUND: Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. METHODS: Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia-telangiectasia mutated (ATM). RESULTS: Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. CONCLUSIONS: The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

Role of a novel benzoxazine derivative in the chemosensitization of colon cancer.

The concept to fight against tumour resistance is to use chemosensitizers that selectively sensitize tumour cells to chemotherapeutic drugs without affecting normal tissue. In this study, the chemosensitizing potential of a novel benzoxazine derivative in combination with Doxorubicin, a DNA damaging chemotherapeutic drug was evaluated. The results of this study showed that the compound LTUR6 is a potent chemosensitizer of Doxorubicin in colon cancer cell lines, HCT116 and HT29. It was also observed that LTUR6 delayed the resolution of Doxorubicin-induced γH2AX, a specific marker of unrepaired DNA DSB, and prolonged cell cycle arrest in both cell lines. This eventually led to DNA fragmentation, caspase activation and ultimately apoptosis in LTUR6 treated cell lines. Results of western blot analysis revealed that LTUR6 significantly inhibited the phosphorylation of DSB repair enzyme AKT, in response to Doxorubicin-induced DSB. We propose that the chemosensitization observed following inhibition of PI3K is likely due to the involvement of a number of downstream targets of AKT.

Student perceptions and learning outcomes of blended learning in a massive first-year core physiology for allied health subjects.

Evidence shows that factors contributing to success in physiology education for allied health students at universities include not only their high school achievement and background but also factors such as confidence with their teachers and quality of their learning experience, justifying intensive and continued survey of students' perceptions of their learning experience. Here we report data covering a 3-yr period in a physiology subject that has been redesigned for blended and online presentation. Consistent with previous reports, we show that when we undertook a blended mode of delivery, students demonstrated better grades than traditional modes of teaching; however the absence of didactic teaching in this subject resulted in lower grades overall. Students have very strong positive attitudes to weekly quizzes (80% positive approval) but report ambivalent attitudes to online self-directed learning (61% negative perception), even though they had 2-h weekly facilitated workshops. Overwhelmingly, students who undertook the subject in a self-directed online learning mode requested more face-to-face-teaching (70% of comments). From these data, we suggest that there is a quantifiable benefit to didactic teaching in the blended teaching mode that is not reproduced in online self-directed learning, even when face-to-face guided inquiry-based learning is embedded in the subject.

Novel benzoxazines as inhibitors of angiogenesis.

Dysregulation of angiogenesis has been associated with many pathological disorders, including cancer; where angiogenesis has been found to be critical for the maintenance and metastasis of tumours. One of the pathways involved in the regulation of angiogenesis is the phosphatidylinositol3-kinase (PI3K) signalling pathway. The PI3K family consists of enzymes that phosphorylate the 3-OH of the inositol ring of phosphatidyl inositol. There are four isoforms, PI3Kα, PI3Kß, PI3Kγ and PI3Kδ, that are signalling intermediaries involved in numerous pathways that sustain and maintain the tumours. In this study, we screened eight novel benzoxazine inhibitors of both PI3K isoforms and the related DNA-PK, for their anti-angiogenic effects. Our findings identified the novel benzoxazine (7, 8 (substituted)-2-morpholino-benz (1,3) oxazine: LTUSI122) to be non-toxic at concentrations up to 5 µM, while exhibiting significant inhibition of various aspects of angiogenesis including endothelial proliferation, migration and tube formation. The molecular mechanisms were examined using an angiogenesis array, revealing inhibition of several proliferative and migratory angiogenic factors, including VEGFR, MMP, IL-8, uPAR and MCP; and stimulation of the endogenous inhibitor, endostatin. We hypothesize that these anti-angiogenic effects are mediated by targeting an important signaling intermediary, PI3Kα, and subsequently its action on vascular endothelial growth factor (VEGF, a key growth factor in the process of angiogenesis). If used in combination with more targeted therapies, LTUSI122 could reduce tumour growth and increase the efficacy of these treatments.

Radiosensitizing activity of a novel Benzoxazine through the promotion of apoptosis and inhibition of DNA repair.

The DNA dependant protein kinase (DNA-PK) enzyme plays a major part in the repair of double stranded breaks induced by radiation and hence in the radio-resistance of tumour cells. Inhibitors of DNA-PK have been tested successfully in the past for their ability to sensitize cancer cells to the effects of radiation. Here we present a novel benzoxazine, 8-methyl-2-(morpholine-4yl)-7-(pyridine-3-methoxy)-4H-1,3-benzoxacine-4-one (LTU27) and analyse its ability to cause sensitization of lung cancer and colon cancer cells to radiation. There was a significant reduction in survival rate, increase in apoptosis and inhibition in autophosphorylation of DNA-PK and AKT1 after treating them concomitantly with both radiation and LTU27. The mechanism of action appears to be through inhibition of DNA-PK leading to delayed DNA repair and promotion of apoptosis.

Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

The benzoaxines have been developed from structurally similar chromones as specific inhibitors of the PI3K family to sensitize cancer cells to the effects of chemotherapeutic agents; most have been shown to do this through specific inhibition of DNA-PK and DNA repair mechanisms. In this study we examined the benzoxazine, 2-((3-methoxybut-3en-2-yl)amino)-8methyl-4H-benzo[1,3]oxazin-4one (LTUSI54). This compound had no DNA-PK or PI3K inhibitory activity but still sensitized HeLa cells to the effects of Etoposide. LTUSI54 works synergistically with Etoposide to inhibit growth of HeLa cells and sub G1 analysis indicates that this is not due to an increase in apoptosis. LTUSI54 neither enhances DSB formation due to Etoposide nor does it delay the repair of such damage. Cell cycle analysis shows a clear G2 block with Etoposide alone while, in combination with LTUSI54 there is an additional S phase arrest. Phospho-kinase analysis indicated that LTUSI54 engages key regulators of cell cycle progression, specifically p38α, p53 and ERK 1/2. From our results we hypothesize that LTUSI54 is promoting the cell cycle arrest through activation of p38α pathways, independent of p53 mechanisms. This results in a decrease in p53 phosphorylation and hence, restricted apoptosis. Changes in cell number appear to be the result of p38α pathways disrupting cell cycle progression, at the S and G2 checkpoints. Further investigation into the finer mechanisms by which LTUSI54 effects cell cycle progression would be of great interest in assessing this compound as a chemosensitising agent.

A systematic review and meta-analyses of interleukin-1 receptor associated kinase 3 (IRAK3) action on inflammation in in vivo models for the study of sepsis.

BACKGROUND: Interleukin-1 receptor associated kinase 3 (IRAK3) is a critical modulator of inflammation and is associated with endotoxin tolerance and sepsis. Although IRAK3 is known as a negative regulator of inflammation, several studies have reported opposing functions, and the temporal actions of IRAK3 on inflammation remain unclear. A systematic review and meta-analyses were performed to investigate IRAK3 expression and its effects on inflammatory markers (TNF-α and IL-6) after one- or two-challenge interventions, which mimic the hyperinflammatory and immunosuppression phases of sepsis, respectively, using human or animal in vivo models. METHODS: This systematic review and meta-analyses has been registered in the Open Science Framework (OSF) (Registration DOI: 10.17605/OSF.IO/V39UR). A systematic search was performed to identify in vivo studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data was available. RESULTS: The search identified 7778 studies for screening. After screening titles, abstracts and full texts, a total of 49 studies were included in the systematic review. The review identified significant increase of IRAK3 mRNA and protein expression at different times in humans compared to rodents following one-challenge, whereas the increases of IL-6 and TNF-α protein expression in humans were similar to rodent in vivo models. Meta-analyses confirmed the inhibitory effect of IRAK3 on TNF-α mRNA and protein expression after two challenges. CONCLUSIONS: A negative correlation between IRAK3 and TNF-α expression in rodents following two challenges demonstrates the association of IRAK3 in the immunosuppression phase of sepsis. Species differences in underlying biology affect the translatability of immune responses of animal models to human, as shown by the dissimilarity in patterns of IRAK3 mRNA and protein expression between humans and rodents following one challenge that are further influenced by variations in experimental procedures.

Lytic Bacteriophage EFA1 Modulates HCT116 Colon Cancer Cell Growth and Upregulates ROS Production in an Enterococcus faecalis Co-culture System.

Enterococcus faecalis is an opportunistic pathogen in the gut microbiota that's associated with a range of difficult to treat nosocomial infections. It is also known to be associated with some colorectal cancers. Its resistance to a range of antibiotics and capacity to form biofilms increase its virulence. Unlike antibiotics, bacteriophages are capable of disrupting biofilms which are key in the pathogenesis of diseases such as UTIs and some cancers. In this study, bacteriophage EFA1, lytic against E. faecalis, was isolated and its genome fully sequenced and analyzed in silico. Electron microscopy images revealed EFA1 to be a Siphovirus. The bacteriophage was functionally assessed and shown to disrupt E. faecalis biofilms as well as modulate the growth stimulatory effects of E. faecalis in a HCT116 colon cancer cell co-culture system, possibly via the effects of ROS. The potential exists for further testing of bacteriophage EFA1 in these systems as well as in vivo models.

Analysis of interleukin-1 receptor associated kinase-3 (IRAK3) function in modulating expression of inflammatory markers in cell culture models: A systematic review and meta-analysis.

BACKGROUND: IRAK3 is a critical modulator of inflammation in innate immunity. IRAK3 is associated with many inflammatory diseases, including sepsis, and is required in endotoxin tolerance to maintain homeostasis of inflammation. The impact of IRAK3 on inflammatory markers such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell culture models remains controversial. OBJECTIVE: To analyse temporal effects of IRAK3 on inflammatory markers after one- or two-challenge interventions in cell culture models. METHODS: A systematic search was performed to identify in vitro cell studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data were available. Comparisons of outcome measures were performed between different cell lines and human and mouse primary cells. RESULTS: The literature search identified 7766 studies for screening. After screening titles, abstracts and full-texts, a total of 89 studies were included in the systematic review. CONCLUSIONS: The review identifies significant effects of IRAK3 on decreasing NF-κB DNA binding activity in cell lines, TNF-α protein level at intermediate time intervals (4h-15h) in cell lines or at long term intervals (16h-48h) in mouse primary cells following one-challenge. The patterns of TNF-α protein expression in human cell lines and human primary cells in response to one-challenge are more similar than in mouse primary cells. Meta-analyses confirm a negative correlation between IRAK3 and inflammatory cytokine (IL-6 and TNF-α) expression after two-challenges.

Teaching mode efficiency and learning preferences of first year nursing students.

The student population in universities is very diverse: ranging ages, experiences, culture, level of preparedness and learning styles. This diversity presents academics with increasing challenges to motivate and promote student understanding. The aim of the current study was to develop knowledge of different learning styles among first year health science students and determine the benefits that students obtain from each teaching strategy. A questionnaire was designed for quantitative data collection, consisting of two sections. The first section sought student feedback on their experiences of lectures, tutorials and practical classes; the second section, consists of the 'VARK test', Visual, Aural, Read/write, Kinesthetic sensory modalities determines the different ways of receiving information [Fleming, N.D., 1995. I'm different; not dumb. Modes of presentation (VARK) in the tertiary classroom. In: Zelmer, A. (Ed.), Annual Conference of the Higher Education and Research Development Society of Australasia. J.]. The study identified that the majority of students found the lectures, tutorials and practical sessions to be beneficial to their learning and the combination reiterates and emphasises various life science concepts. The most favoured strategy was practical sessions, while tutorials were seen as least useful. The sensory mode the majority of students preferred to receive information was kinesthetic, the hands on approach to learning. Students are diverse creatures with differing abilities and mode for learning. There is no single right way to present material but by providing several different approaches the differing learning styles of students can be accommodated.

Determination of the DNA repair pathways utilised by acute lymphoblastic leukaemia cells following daunorubicin treatment.

OBJECTIVE: DNA double strand breaks (DNA-DSBs) are among the most lethal DNA lesions leading to genomic instability and repaired by either homologous recombination (HR) or the non-homologous end joining (NHEJ) mechanisms. The purpose of this study was to assess the importance and the level of activation of non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways in three cell lines, CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes following treatment with chemotherapy agent daunorubicin. RESULTS: The Gamma histone H2AX (γH2AX) assay was used assess the effects of DNA-PK inhibitor NU7026 and RAD51 inhibitor RI-2 on repair of DNA-DSB following treatment with daunorubicin. In all cell lines, the NHEJ DNA repair pathway appeared more rapid and efficient. MOLT-4 and CCFR-CEM cells utilised both NHEJ and HR pathways for DNA-DSB repair. Whereas, SUP-B15 cells utilised only NHEJ for DSB repair, suggestive of a deficiency in HR repair pathways.

"There's no point in complaining, nothing changes": rural disaffection with complaints as an improvement method.

OBJECTIVE: To validate earlier findings that lack of access to health services is the most likely issue of complaint by rural consumers, and that lack of knowledge about how to make effective complaints and scepticism that responses to complaints bring about service improvement account for the under-representation of complaints from rural consumers. DESIGN: Unaddressed reply-paid mail survey to 100% of households in small communities, and 50%, 20% or 10% in progressively larger communities. SETTING: Eight communities in the Loddon-Mallee region of Victoria. PARTICIPANTS: 983 householders most responsible for the health care of household members, responding to a mailed questionnaire. MAIN OUTCOME MEASURES: Issues of complaints actually made; issues of unsatisfactory situations when a complaint was not made; reasons for not complaining; to whom complaints are made; and plans for dealing with any future complaint. RESULTS: Earlier findings were confirmed. Lack of access to health services was the most important issue, indicated by 54.8% of those who had made a complaint, and 72% of those who wanted to but did not. The most common reason given for not complaining was that it was futile to do so. Lack of knowledge of how to make effective complaints which might contribute to the quality assurance cycle was evident. CONCLUSIONS: Rural consumers' disaffection with health complaints as a means to quality improvement poses a significant barrier to consumer engagement in quality assurance processes. Provider practices may need to change to regain community confidence in quality improvement processes.

How do rural consumers contribute to quality assurance of health services?

OBJECTIVE: To investigate the reasons for complaint or non-complaint by rural consumers of health services. DESIGN: Qualitative study using focus group discussion of hypothetical scenarios. SETTING: Selected rural communities in the Loddon-Mallee region of north-western Victoria. PARTICIPANTS: Sixty volunteer participants in eight focus groups recruited through advertising. MAIN OUTCOME MEASURE: Issues and themes concerning circumstances leading to, and factors inhibiting, complaints about a health service and awareness of complaints mechanisms. RESULTS: Compared with residents of larger towns, those of small communities were more likely to report they would complain to the local provider, whereas those in larger towns were more likely to mention Hospital Boards or the Commissioner. Deterrents to making complaints included the lack of services, scepticism about the role of complaints in bringing about change and an attitude that it was more appropriate to try to fix the problem than complain about it. Lack of awareness of appropriate complaint mechanisms which feed into quality assurance processes was also identified. CONCLUSIONS: Previously documented lower complaint rates from rural consumers can not be taken to mean greater satisfaction with health services.

The experience of general nurses in rural Australian emergency departments.

Australia is a geographically unique country with large areas classed as rural. Nurses providing emergency care in rural hospitals face a number of challenges, with rural communities expecting multi-skilled nurses, prepared for a wide range of unannounced situations. Using a mixed method approach, involving questionnaires and focus groups, the study was undertaken in two rural health services in Victoria, Australia. The aim was to explore the experiences of general nurses working in rural hospital settings, with regards to their emergency department responsibilities. The findings indicate that nurses lacked confidence, which they attributed to the sporadic nature of working in the area and the diversity of people who presented. A resultant 'skills rusting' was described and nurses identified the need to be a diverse 'specialist'. Some lack of confidence, particularly in the mental health area, was related to feelings of isolation and lack of context specific education and training. While some excellent emergency specific education and training is available for rural nurses, access is limited by a multitude of constraints. This study found there is an urgent need for local emergency education and training; with nurses showing a strong preference for ongoing professional development incorporating scenario based and context specific education.