Young people's Perception of Group Climate in Juvenile Justice Centers in an Australian State, a Pilot Study.

There is ample evidence to demonstrate that a positive group climate in juvenile justice centers contributes to improvements in motivation for positive behavior and reducing incidents and even recidivism. Being provided opportunities for growth and development and being actively supported by well-trained staff creates a positive atmosphere and minimal repression. Such a pedagogical group climate is strongly related to fulfilling the basic psychological needs of the young people and is promoted by relational security. Following a series of challenging situations inside youth justice centers in Australia, we explored the opportunities for investing in staff training on awareness of basic psychological needs of these young people and introducing the concept of an open, positive group climate. This study reports the results of an explorative measurement of perceived group climate in four units of two juvenile justice centers in Victoria (Australia). The young people (N = 57) were provided with the Prison Group Climate Instrument. The results of the group climate research and a cross-cultural comparison show that there are inherent strengths but also important opportunities for improvement in the group climate experienced by young people. Recommendations to improve the group climate are discussed in this paper.

The burden of Legionnaires' disease in New Zealand (LegiNZ): a national surveillance study.

BACKGROUND: Legionnaires' disease is under-diagnosed because of inconsistent use of diagnostic tests and uncertainty about whom to test. We assessed the increase in case detection following large-scale introduction of routine PCR testing of respiratory specimens in New Zealand. METHODS: LegiNZ was a national surveillance study done over 1-year in which active case-finding was used to maximise the identification of cases of Legionnaires' disease in hospitals. Respiratory specimens from patients of any age with pneumonia, who could provide an eligible lower respiratory specimen, admitted to one of 20 participating hospitals, covering a catchment area of 96% of New Zealand's population, were routinely tested for legionella by PCR. Additional cases of Legionnaires' disease in hospital were identified through mandatory notification. FINDINGS: Between May 21, 2015, and May 20, 2016, 5622 eligible specimens from 4862 patients were tested by PCR. From these, 197 cases of Legionnaires' disease were detected. An additional 41 cases were identified from notification data, giving 238 cases requiring hospitalisation. The overall incidence of Legionnaires' disease cases in hospital in the study area was 5·4 per 100 000 people per year, and Legionella longbeachae was the predominant cause, found in 150 (63%) of 238 cases. INTERPRETATION: The rate of notified disease during the study period was three-times the average over the preceding 3 years. Active case-finding through systematic PCR testing better clarified the regional epidemiology of Legionnaires' disease and uncovered an otherwise hidden burden of disease. These data inform local Legionnaires' disease testing strategies, allow targeted antibiotic therapy, and help identify outbreaks and effective prevention strategies. The same approach might have similar benefits if applied elsewhere in the world. FUNDING: Health Research Council of New Zealand.

Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells.

A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression, without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.

Transformation and removal of wood extractives from pulp mill sludge using wet oxidation and thermal hydrolysis.

In order to remove wood extractive compounds from pulp mill sludge and thereby enhancing anaerobic digestibility, samples were subjected to either oxidative hydrothermal treatment (wet oxidation) or non-oxidative hydrothermal treatment (thermal hydrolysis). Treatments were carried out at 220 °C with initial pressure of 20 bar. More than 90% destruction of extractive compounds was observed after 20 min of wet oxidation. Wet oxidation eliminated 95.7% of phenolics, 98.6% fatty acids, 99.8% resin acids and 100% of phytosterols in 120 min. Acetic acid concentration increased by approximately 2 g/l after 120 min of wet oxidation. This has potential for rendering sludge more amenable to anaerobic digestion. In contrast thermal hydrolysis was found to be ineffective in degrading extractive compounds. Wet oxidation is considered to be an effective process for removal of recalcitrant and inhibitive compounds through hydrothermal pre-treatment of pulp mill sludge.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

Development of second-generation VEGFR tyrosine kinase inhibitors: current status.

The vascular endothelial growth factor (VEGF) signaling pathway appears to be the dominant pathway involved in tumor angiogenesis, providing a rationale for targeting the VEGF receptors (VEGFR-1, -2, and -3) in the treatment of cancers. In particular, VEGF signaling is thought to be important in renal cell carcinoma (RCC) because of the deregulation of the pathway through nearly uniform loss of the von Hippel Lindau protein. The tyrosine kinase inhibitors (TKIs) sorafenib, sunitinib, and pazopanib are approved by the US Food and Drug Administration for the treatment of advanced RCC; however, these multitargeted agents inhibit a wide range of kinase targets in addition to the VEGFRs, resulting in a range of adverse effects unrelated to efficient VEGF blockade. This article reviews recent advances in the development of the second-generation VEGFR TKIs, including the more selective VEGFR TKIs tivozanib and axitinib, and focuses on the potential benefits of novel inhibitors with improved potency and selectivity.

Chimeric mouse tumor models reveal differences in pathway activation between ERBB family- and KRAS-dependent lung adenocarcinomas.

To recapitulate the stochastic nature of human cancer development, we have devised a strategy for generating mouse tumor models that involves stepwise genetic manipulation of embryonic stem (ES) cells and chimera generation. Tumors in the chimeric animals develop from engineered cells in the context of normal tissue. Adenocarcinomas arising in an allelic series of lung cancer models containing HER2 (also known as ERBB2), KRAS or EGFR oncogenes exhibit features of advanced malignancies. Treatment of EGFR(L858R) and KRAS(G12V) chimeric models with an EGFR inhibitor resulted in near complete tumor regression and no response to the treatment, respectively, accurately reflecting previous clinical observations. Transcriptome and immunohistochemical analyses reveal that PI3K pathway activation is unique to ERBB family tumors whereas KRAS-driven tumors show activation of the JNK/SAP pathway, suggesting points of therapeutic intervention for this difficult-to-treat tumor category.

De novo discovery of a gamma-secretase inhibitor response signature using a novel in vivo breast tumor model.

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.

Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice.

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

A telomerase enzymatic assay that does not use polymerase chain reaction, radioactivity, or electrophoresis.

A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.

Functional conservation of the telomerase protein Est1p in humans.

Eukaryotic telomerase contains a telomerase reverse transcriptase (TERT) and an RNA template component that are essential for telomerase catalytic activity and several other telomerase-associated factors of which only a few appear to be integral enzyme components [1-3]. The first essential telomerase protein identified was S. cerevisiae Est1p, whose deletion leads to ever-shorter telomeres despite the persistence of telomerase activity [4-6]. Extensive genetic and biochemical data show that Est1p, via its interaction with the telomerase RNA and telomere end DNA binding complex Cdc13p/Stn1p/Ten1p, promotes the ability of telomerase to elongate telomeres in vivo [7-22]. The characterization of Est1p homologs outside of yeast has not been documented. We report the characterization of two putative human homologs of Est1p, hEST1A and hEST1B. Both proteins specifically associated with telomerase activity in human cell extracts and bound hTERT in rabbit reticulocyte lysates independently of the telomerase RNA. Overproduction of hEST1A cooperated with hTERT to lengthen telomeres, an effect that was specific to cells containing telomerase activity. Like Est1p, hEST1A (but not hEST1B) exhibited a single-stranded telomere DNA binding activity. These results suggest that the telomerase-associated factor Est1p is evolutionarily conserved in humans.

Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert.

Prolonged growth of murine embryonic stem (ES) cells lacking the telomerase reverse transcriptase, mTert, results in a loss of telomere DNA and an increased incidence of end-to-end fusions and aneuploidy. Furthermore, loss of only one copy of mTert also results in telomere shortening intermediate between wild-type (wt) and mTert-null ES cells [Liu, Y., Snow, B. E., Hande, M. P., Yeung, D., Erdmann, N. J., Wakeham, A., Itie, A., Siderovski, D. P., Lansdorp, P. M., Robinson, M. O. & Harrington, L. (2000) Curr. Biol. 10, 1459-1462]. Unexpectedly, although average telomere length in mTert(+/-) ES cells declined to a similar level as mTert-null ES cells, mTert(+/-) ES cell lines retained a minimal telomeric DNA signal at all chromosome ends. Consequently, no end-to-end fusions and genome instability were observed in the latest passages of mTert(+/-) ES cell lines. These data uncover a functional distinction between the dosage-dependent function of telomerase in average telomere-length maintenance and the selective maintenance of critically short telomeres in cells heterozygous for mTert. In normal and tumor cells, we suggest that telomerase activity insufficient to maintain a given average telomere length may, nonetheless, provide a protective advantage from end-to-end fusion and genome instability.

Telomere dysfunction: multiple paths to the same end.

The molecular cloning of telomerase and telomere components has enabled the analysis and precise manipulation of processes that regulate telomere length maintenance. In mammalian cells and in other organisms, we now recognize that disruption of telomere integrity via any one of a number of perturbations induces chromosome instability and the activation of DNA damage responses. Thus, telomere dysfunction may represent a physiological trigger of the DNA damage or apoptotic response in an analogous fashion to other genotoxic insults that introduce chromosome breaks. Initial studies in mice lacking the murine telomerase RNA and in cells expressing a dominant negative version of the telomere binding protein TRF2 revealed a strong p53-dependent response to telomere dysfunction. Yet, telomere dysfunction exhibits p53-independent effects as well, an observation supported by p53-independent responses to telomere dysfunction in p53 mutant human tumor cell lines and mouse cells. As most tumors are compromised for p53 function, examination of this p53-independent response warrants closer attention. A better understanding of this p53-independent response may prove critical for determining the ultimate utility of telomerase inhibitors in the clinic. This review will summarize our current understanding of the molecular responses to telomere dysfunction in mammalian cells.