The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and β-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

Identification of genes differentially regulated by the P210 BCR/ABL1 fusion oncogene using cDNA microarrays

Objective: The t(9;22) translocation is associated with more than 95% of cases of chronic myeloid leukemia. The resulting fusion of the BCR and ABL1 loci produces the constitutively active BCR/ABL1 tyrosine kinase. A wide range of signal transduction molecules are activated by BCR/ABL1, including MYC, PI-3 kinase, and different STAT molecules. In contrast, relatively few genes are known to be regulated by BCR/ABL1 at the level of transcription.

Materials and Methods: In an effort to better understand the transcriptional program activated by BCR/ABL1, we used cDNA microarrays to evaluate the relative expression of approximately 6450 human genes in U937 myelomonocytic cells expressing P210 BCR/ABL1 via a tetracycline-inducible promoter.

Results: We confirmed the previously reported up-regulation of the PIM1 and JUN oncogenes by BCR/ABL1. In addition, we identified 59 more genes up-regulated by BCR/ABL1. Interestingly, roughly one third of these were genes previously reported to be interferon (IFN)-responsive, including the OAS1, IFIT1, IFI16, ISGF3G, and STAT1 genes. An additional seven BCR/ABL1-regulated genes were found to be IFN-responsive in U937 cells. The expression profile also included genes encoding transcription factors, kinases, and signal transduction molecules, as well as genes regulating cell growth, differentiation, apoptosis, and cell adhesion, features previously suggested to be affected by BCR/ABL1.

Conclusion: These observations shed novel insight into the mechanism of BCR/ABL1 action and provide a range of targets for further investigation.

Recent advances on the possible neuroprotective activities of Epstein-Barr virus oncogene BARF1 protein in chronic inflammatory disorders of central nervous system

Multiple sclerosis and neurodegenerative diseases in which cells of the central nervous system (CNS) are lost or damaged are rapidly increasing in frequency, and there is neither effective treatment nor cure to impede or arrest their destructive course. The Epstein-Barr virus is a human gamma-herpesvirus that infects more than 90% of the human population worldwide and persisting for the lifetime of the host. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. Individuals with a history of symptomatic primary EBV infection, called infectious mononucleosis, carry a moderately higher risk of developing multiple sclerosis (MS). It is not known how EBV infection potentially promotes autoimmunity and central nervous system (CNS) tissue damage in MS. Recently it has been found that EBV isolates from different geographic regions have highly conserved BARF1 epitopes. BARF1 protein has the neuroprotective and mitogenic activity, thus may be useful to combat and overcome neurodegenerative disease. BARF1 protein therapy can potentially be used to enhance the neuroprotective activities by combinational treatment with anti-inflammatory antagonists and neuroprotectors in neural disorders.

Implementing elements of Schon's reflective practitioner in an undergraduate public relations program

While the internship unit in a public relations degree is often viewed as the foremost area of experiential learning it can fail to deliver significant learning outcomes for student and teacher. This paper explores the use of other activity-based learning (ABL) methods as implemented in the Deakin University public relations undergraduate degree. The paper applies Donald Schon's concept of the reflective practitioner to learning undertaken in an internship unit and compares this application to two other units where ABL is used and the focus is on creating a client-professional relationship. For the purpose of this paper information was analysed using the 2006 on-campus cohort only. The cohort was further refined to include those students who successfully completed the degree of BA in public relations. Student internship evaluation forms and unit records were viewed to gain statistical information.

Nutrition, growth and production of Cherax Destructor Clark, 1936 (Decapoda, Parastacidae)

In this study the nutrition, growth and production of C. destructor was examined. Selected nutritional requirements of juvenile animals were determined under controlled conditions with the aim of developing a pelleted diet for use in hatcheries, nurseries and growout situations. The best developed diet was assessed for its potential as a supplementary feed for animals cultured in earthen environments. The protein requirements were first determined simultaneously with an evaluation of the effect of replacing animal protein (fishmeal) by soybean meal. Juveniles were reared communally for 59 d on isoenergetic diets containing 15-30% protein and graded levels of soybean meal (0-60%, of protein). When soybean meal was included at a level of 40-60%, growth was reduced relative to that achieved with control diets containing 15% and 20% protein, but this was not the case at a 20% soybean meal substitution level. A two-way interaction occurred between dietary protein and soybean meal content. Higher protein feeds enabled higher soybean meal inclusion levels without significantly affecting growth. Protein increases of 5% produced better growth at the 40% and 60% soybean meal substitution levels. This effect was less pronounced in the control and the 20% soybean meal diets. Carcass %protein increased and %lipid decreased as dietary protein increased. A similar effect occurred by increasing the soybean meal level to 60%. No obvious trend in carcass moisture, energy, and ash occurred. A protein requirement of 30% was apparent when fish meal and soybean meal were included in diets at levels of 20% and 24% (dry matter) respectively. Alternative protein sources to soybean meal were subsequently identified. Juveniles were maintained for 12 weeks on isoenergetic diets containing 30% protein and differing in the primary source of protein used, with meat, snail, soybean, yabby, and zooplankton meals comprising the major protein ingredient. No significant difference occurred in mean weight (MW), percentage weight gain (%WG), SGR or survival among diets. Food conversion ratios (FCR) were low, with a minimum value of 0.95 for the snail-based diet. The apparent net protein utilisation (ANPU) varied from 29.6% (zooplankton-based diet) to 41.2% (snail-based diet). Carcass composition varied with diet, with the greatest difference occurring in carapace colour. Animals fed the zooplankton-based diet developed the strongest, most natural pigmentation. A new combination of previously used protein-based ingredients was subsequently tested with reference to two yabby species, Cherax albidus and Cherax destructor, that were grown simultaneously in identical conditions. Juvenile male animals were reared individually for 20 weeks on isoenergetic diets containing 15% or 30% protein with fish meal, soybean meal, yabby meal and wheat products forming the basis of the diets. C albidus grew the fastest and utilised the food the most effectively. Carcass composition was influenced by diet with the 30% protein diet resulting in an increase in carcass protein and ash and a decrease in carcass lipid and energy relative to the low protein diet. Carcass moisture and calcium were not affected by diet. The intermoult period (IP) was highly dependent on the premoult weight (W) but the mean moult increment (WI, as weight) was independent of the PM. The orbital carapace length (OCL) and the abdominal length (ABL) %moult increments generally declined with an increase in PM whereas the propus length (PL) %moult increment generally increased. The IP, WI, %OCL, %ABL, and %PL moult increments varied according to diet and to species. Elevated dietary protein caused a reduction to the IP (for similar sized animals) by 11 d and 7 d and an increase to the WI by 85% and 81% in C. albidus and C destructor respectively. Dietary induced morphological changes also occurred. Animals of a standard OCL (both species) had significantly larger abdomens when fed the higher protein diet. Growth on the best developed diet was compared to the growth obtained on a natural diet of freshwater zooplankton. Juveniles were reared individually for 12 weeks on the two diets. The MW, %WG and SGR were higher for the zooplankton diet. Carcass composition was influenced by diet and the zooplankton fed animals had a higher carcass %protein, %lipid, %ash and %fibre content and were more richly pigmented than animals fed pellets. The IP and the WI were highly dependent on the PM and varied according to diet; feeding with zooplankton reduced the IP by 1.2 days and increased the WI by 13.7% compared to pellets. Nutrient digestibility was determined for the pelleted diets evaluated in the growth trials. Protein digestibility (PD) and dry matter digestibility (DMD), using chromic oxide (Cr2O3) as an exogenous marker, were high for all diets, at around 93% and 83% respectively. Ash digestibility varied considerably from 17% to 73% for the snail and yabby meal diets respectively. Crude fibre digestibility was around 50% and probably indicates cellulase activity. Alternative markers to Cr2O3 were evaluated. Ash was considered to be the most suitable alternative to Cr2O3, providing a reasonable, albeit lower, estimate of nutrient digestibility. Cr2O3 and ash were preferentially excreted whereas fibre was retained in the digestive system for a longer period, consequently, the collection of a particular fraction of the deposited faeces (late or early) substantially affected the digestibility coefficients. In earthen-based environments, animals fed the best developed diet were compared to animals cultured using a forage crop of clover (Trifolium repens). Three supplementary feeding strategies representing varying levels of management intensity were evaluated in a series of trials conducted in ponds and pond microcosms. Growth on pellets consistently exceeded that obtained with the forage crop, with final MW being 67-159% higher than that using clover and appeared to be the result of direct pellet consumption and from a pellet fertiliser effect (on the sediment). Within-pond DMD and PD were high and similar for each treatment (DMD = 51-58%; PD = 89-92%). In the control pond, DMD and PD increased with each successive flood. The faecal egestion rate (PER) decreased with each successive flood in all ponds, and is negatively related to animal weight and to foregut fullness (FF) according to power curves. FF was consistently lowest in the control pond. Mean FF was 48.5%, 62.3%, and 26.7% for the pellet, crop and control ponds respectively. FF increased to the third flood in each pond. The foregut protein content was high in all samples and the mean values were 33.9%, 32.7% and 35.6% for the pellet, crop and control ponds respectively. Foregut ash was highly variable within each pond and is inversely related to the foregut protein content. In the control and pellet ponds the highest foregut ash content occurred during flood 1. The culture system (aquaria or pond) strongly influenced the composition of the foregut content. The foregut of animals fed the manufactured diet (B2) in ponds contained approximately 176% more ash and 5% more protein than the foregut of animals fed in bare-bottom tanks. The FF of the tank fed animals was approximately 45% higher than the FF of pond fed animals after a similar feeding period. Base-line yields for extensive production systems appeared to be around 400kg ha-1. The supplementary addition of T. repens produced yields of approximately 635kg ha-1 (in ponds) to around 1086kg ha-1 (in tanks). The sequential addition of cut-clover to tanks stimulated growth to levels approaching those achieved on pellets. Yabbies stocked into ponds at 15-20 m-2 with a mean weight of 2.67g and fed a 30% protein pelleted diet for 100 d, resulted in a yield of approximately 1117kg ha-1, but only 2% of the population were above a marketable size of 50g. The feed utilisation indices were better for animals reared on pellets in bare-bottom tanks than in earthen environments, indicating some degree of pellet wastage when natural feeds are simultaneously present. High apparent food conversion ratios and low protein efficiency ratios occurred when the forage crop was provided. A considerable quantity of the dry matter and protein content of the forage crop was either inefficiently utilised or directed into other production pathways. Sowing a forage crop into pond microcosms to which a pelleted diet was also provided, did not enhance growth performance. Pelleted feed inputs at a rate of approximately 129g m-2 to 198g m-2 (dry matter) and 38g -2 to 64g m-2 (protein) over 70-100 d resulted in acceptable growth and feed utilisation indices for animals reared in ponds and pond microcosms. Forage crop inputs of approximately 533g m-2 to 680g m-2 (as dry matter) or 84g m-2 to 177g m-2 (as protein) over a 70-100 d period produced reasonable growth rates but poor feed utilisation indices. Low inputs of dry matter (from 113-296g m-2) and protein (from 24-54g m-2) from clover were sufficient to maintain high growth rates in pond microcosms for around 28 d. In ponds, a very low level of 21g m-2 (dry matter) and 4.3g m-2 (protein) was sufficient for around 3 weeks. Forage depletion appeared to occur beyond week 3-4 and was probably a major growth limiting factor. The mean hepatosomatic index (HSI) was 9.44, 7.68, and 6.79 for the pellet, crop, and control ponds respectively. The relationship between hepatopancreas weight and overall animal weight was significantly different between treatments. The hepatopancreas of pellet-fed animals had the highest %lipid and lowest %ash, %protein, %carbohydrate and %moisture content. In terms of absolute quantities, the only major difference in hepatopancreas composition between treatments occurred for lipid and dry matter content. The hepatopancreas of the pellet-fed animals was a cream/cream-yellow colour and was very fragile, whereas in the other ponds it was a more ‘natural’ bright yellow colour and was structurally more robust. C. destructor has a capacious foregut, being approximately 5 times the volume of similar sized Penaeids. The foregut volume (V, ml) of the yabby is related to animal weight (W, g) according to V = 0.048 W0.9543. Animals that were starved for 96 h and then fed diet B2 were almost completely foil after 30 min. The ‘apparent enzymatic response’ of animals fed various natural and artificial diets in tanks was evaluated. Nutrient processing time and the enzymatic response following ingestion appeared to be regulated by the chemical and physical properties of the diet. For the natural feeds, foregut protein was 1.2% higher (for zooplankton) and up to 300% higher (for detritus) than dietary protein, whereas ash was 7.5% higher (zooplankton) and 46-63% lower (detritus) than dietary ash. For animals fed diet B2 after 48 h without food, FF was approximately half that of 96 h starved animals after a similar feeding period but foregut protein and ash contents were similar. Finally, the physiological and morphological attributes elucidated in this study are discussed with reference to the ecology of the yabby. High growth rates, excellent feed utilisation indices and high digestibility coefficients for a wide range of diet-types illustrate nutritional flexibility. A capacious foregut, a large hepatopancreas with a high energy storage capacity, the ability to partition and preferentially excrete the low nutrient value inorganic component of the diet, the capacity to alter body form, nutrient processing time and enzymatic secretions in relation to diet-type, and modified behaviour according to feed availability also demonstrate plasticity/adaptability/flexibility. The combined effect of these important characteristics ensures survival in environments that may be adverse and highly variable in terms of nutrient availability. Collectively the morphological and digestive traits elucidated in this study reflect the generalist-type nature of C destructor and indicate that a polytrophic classification still seems appropriate. Several priority areas for further nutrition research are identified and recommendations are made regarding the best-practices to use in the commercial culture of the yabby. Of paramount importance is the further clarification of the nutritional requirements and feeding preferences of animals in various phases of development.

Clinical applications of aptamers and nucleic acid therapeutics in haematological malignancies

Haematological malignancies result from a heterogeneous mix of genetic mutations and chromosome aberrations and translocations. Targeted therapies, such as the anti-CD20 antibody rituximab, or the BCR-ABL1 inhibitor imatinib, have proven to be effective treatments in the management of some of these malignancies, though relapsing or refractory disease is still common. Nucleic acid-based therapies have also entered the clinical arena, providing an alternative, complementary approach. The forerunner of these therapies were the antisense oligonucleotides, but their scope has expanded to include short-interfering RNA (siRNA), microRNA, decoy oligonucleotides and aptamers. These can be used either as monotherapeutics, in conjunction with current chemotherapy regimens, or in combination with each other to improve therapeutic efficacy. Not only can these nucleic acid-based therapies silence target genes, they also have the potential of restoring gene function. While challenges remain in delivering effective doses of nucleic acid in vivo, these are steadily being met, suggesting an optimistic future in the treatment of haematological malignancies. This review summarizes the application of nucleic acid-based therapeutics, particularly aptamers, in the diagnosis and treatment of haematological malignancies.

Origins of adaptive immunity

Adaptive immunity, involving distinctive antibody- and cell-mediated responses to specific antigens based on "memory" of previous exposure, is a hallmark of higher vertebrates. It has been argued that adaptive immunity arose rapidly, as articulated in the "big bang theory" surrounding its origins, which stresses the importance of coincident whole-genome duplications. Through a close examination of the key molecules and molecular processes underpinning adaptive immunity, this review suggests a less-extreme model, in which adaptive immunity emerged as part of longer evolutionary journey. Clearly, whole-genome duplications provided additional raw genetic materials that were vital to the emergence of adaptive immunity, but a variety of other genetic events were also required to generate some of the key molecules, whereas others were preexisting and simply co-opted into adaptive immunity.

Peripheral withdrawal recruits distinct central nuclei in morphine-dependent rats

This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis, accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways.

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

Background Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated.

Design and Methods TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro.

Results Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis.

Conclusions This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

More than a game: evaluation report

‘More than a Game’ is a sport-based youth mentoring program developed and implemented by Western Bulldogs in partnership with Newport Islamic Society (NIS), the Australian Federal Police, Victoria Police and Hobsons Bay City Council, with funding from the Attorney General’s Department Building Community Resilience (BCR) grant scheme. The program aimed to develop a community-based resilience model that would use team-based sports to address issues of identity, sense of belonging and cultural isolation amongst young men of Islamic faith, all of which are identified as factors that may promote forms of violent extremism. The program involved 60 young men, aged 15-25, from the Newport Islamic Society in Melbourne’s Western suburbs. The boys were engaged in numerous activities where they were mentored by staff from Western Bulldogs, Victoria Police and Australian Federal Police, who worked in conjunction with community leaders from the Newport Islamic Society.

Through sports-based training, mentoring programs, and community dialogue, ‘More Than a Game’ aimed to develop participants’ leadership, communication, and cross-cultural engagement skills; to identify and facilitate the development of young role models in the community; to enhance greater understanding of the Muslim community in Melbourne’s West, and to foster greater intercultural contact and understanding between participants and other cultural groups. A number of activities were developed and implemented as a part of the program