Local control, safety, and survival following image-guided percutaneous microwave thermal ablation in primary lung malignancy.

AIM: To determine local control, safety, and survival following percutaneous computed tomography (CT)-guided high-power microwave ablation (MWA) in the treatment of primary lung malignancy at a single institution. MATERIAL AND METHODS: From July 2010 to June 2016, 52 patients (mean age 76.3 years, range 55-91 years) with 61 unresectable primary lung cancers of mean diameter 23.8 mm (range 26-55 mm) underwent MWA in 55 ablation sessions. Tumours were diagnosed at biopsy, or positron-emission tomography (PET) avidity (mean SUV max = 10.51) and interval growth. Statistical analysis was performed by Kaplan-Meier modelling and Cox and logistic regression. RESULTS: Local tumour progression (LTP) was diagnosed in six lesions (10%). Median time to local recurrence was 3 months (range 2-14 months). There was a near 12-fold increased odds of local recurrence if the lesion size was >3 cm (95% confidence interval [CI]: 1.84-75.14; p=0.009). The median inpatient stay was 1 day, with no intra-procedural deaths and a 0% 30-day post-ablation mortality rate. Pneumothorax requiring drain was the most serious complication, occurring in 22% (n=12) of patients. Presence of severe emphysema and predicted forced expiratory volume in 1 second (FEV1) of <50% were found to predict future requirement of a drain (odds ratio [OR] 8.17, 95% CI: 1.62-41.37, p=0.01 and OR: 5.14, 95% CI: 1.28-20.68, p=0.02 respectively), when adjusted for age and gender. Tumour size >3 cm had a hazard ratio of 4.37 compared with tumour size ≤3 cm (95% CI: 1.45-13.17, p=0.009) of risk of cancer death at any time, by Cox regression. CONCLUSION: MWA for primary lung malignancy is a safe and effective treatment for primary lung tumours with outcomes that may be comparable to stereotactic body radiation therapy.

Developing research priorities for palliative care of people with intellectual disabilities in Europe: a consultation process using nominal group technique.

BACKGROUND: Empirical knowledge around palliative care provision and needs of people with intellectual disabilities is extremely limited, as is the availability of research resources, including expertise and funding. This paper describes a consultation process that sought to develop an agenda for research priorities for palliative care of people with intellectual disabilities in Europe. METHODS: A two-day workshop was convened, attended by 16 academics and clinicians in the field of palliative care and intellectual disability from six European countries. The first day consisted of round-table presentations and discussions about the current state of the art, research challenges and knowledge gaps. The second day was focused on developing consensus research priorities with 12 of the workshop participants using nominal group technique, a structured method which involved generating a list of research priorities and ranking them in order of importance. RESULTS: A total of 40 research priorities were proposed and collapsed into eleven research themes. The four most important research themes were: investigating issues around end of life decision making; mapping the scale and scope of the issue; investigating the quality of palliative care for people with intellectual disabilities, including the challenges in achieving best practice; and developing outcome measures and instruments for palliative care of people with intellectual disabilities. CONCLUSIONS: The proposal of four major priority areas and a range of minor themes for future research in intellectual disability, death, dying and palliative care will help researchers to focus limited resources and research expertise on areas where it is most needed and support the building of collaborations. The next steps are to cross-validate these research priorities with people with intellectual disabilities, carers, clinicians, researchers and other stakeholders across Europe; to validate them with local and national policy makers to determine how they could best be incorporated in policy and programmes; and to translate them into actual research studies by setting up European collaborations for specific studies that require such collaboration, develop research proposals and attract research funding.

The single laser flow cytometric micronucleus test: a time course study using colchicine and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood.

A single laser flow cytometric procedure to quantify micronucleus frequency in rat and mouse peripheral blood was evaluated. Reticulocytes express the transferrin receptor (also known as the CD71-defined antigen). When combined with a DNA stain, antibodies against this antigen can be used to differentially label and quantify micronucleated reticulocytes. The object of this study was to evaluate the method for rat and mouse peripheral blood using flow cytometry and compare the results obtained between two laboratories (GlaxoWellcome and Litron Laboratories). The compounds selected were the rodent carcinogens colchicine, urethane and acetaldehyde. Colchicine gives a positive response in the rat bone marrow micronucleus assay and an inconclusive result in the rat peripheral blood micronucleus assay. The latter two are both established rat carcinogens readily detected in both the bone marrow and peripheral blood micronucleus assays. In these experiments both rat and mice were treated with either colchicine or urethane and rats alone treated with acetaldehyde. After a single treatment, repeat sampling of peripheral blood was made at 0, 24, 48 and 72 h. Replicate blood samples were obtained and fixed for flow cytometric analysis at both facilities. The micronucleated reticulocyte frequency of each blood sample was determined by analysing 20 000 total reticulocytes per blood sample. The data suggest that the single laser flow cytometric procedure resulted in consistent reticulocyte and micronucleated reticulocyte frequencies between laboratories. Furthermore, these flow cytometric data compare favourably with previously published data.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Leptin and its role in lipid metabolism.

Since the discovery of leptin in 1994, a considerable amount of research has focused on leptin as a central regulator of body weight. In the animal model, research has demonstrated leptin action through hypothalamic centres altering both satiety and energy expenditure. In contrast to animal studies, it is unlikely that leptin functioning in the human system exerts such a profound role in body weight regulation. Human studies suggest that leptin levels are strongly correlated with both percentage fat mass and body mass index, in accordance with the proposed 'lipostatic theory'. Current research suggests the existence of a unique inter-relationship between dietary fat, leptin expression and leptin action within the peripheral system. More specifically, it has been demonstrated that polyunsaturated fatty acid (PUFA) intake influences adipose tissue expression of leptin, and of several lipogenic enzymes and transcription factors. In addition, leptin stimulates triglyceride depletion in white adipose tissue without increasing free fatty acid release, thus favouring fatty acids versus glucose as a fuel source. Recent studies suggest that the reduction in adipose hypertrophy observed with n-3 PUFA-containing fish oil feeding might involve a leptin-specific process. A large amount of evidence supports direct functioning of leptin in peripheral lipid metabolism in vivo and in vitro. It is possible that PUFAs will maintain an efficient level of circulating leptin, thus preventing leptin insensitivity and weight gain. There has been much recent progress in clinical leptin research, from energy expenditure to leptin analogue efficacy; the purpose of the present review is to summarize our current understanding of leptin functioning.

Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations.

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the alpha and beta isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.

Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin.

The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent functions in folding its in vivo substrates, the actins and tubulins. CCT has been loaded with (35)S-beta-actin by in vitro translation in reticulocyte lysate and then subjected to immunoprecipitation with all eight anti-CCT subunit antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between (35)S-beta-actin and isolated CCTalpha, CCTbeta, CCTepsilon, or CCTtheta subunits are observed, suggesting that polar and electrostatic interactions may mediate actin binding to these four CCT subunits. Additionally, a beta-actin peptide array was screened for CCT-binding sequences. Three regions rich in charged and polar amino acid residues, which map to the surface of native beta-actin, are implicated in interactions between actin and CCT. Several of these biochemical results are consistent with the recent cryo-electron microscopy three-dimensional structure of apo-CCT-alpha-actin, in which alpha-actin is bound by the apical domains of specific CCT subunits. A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits.

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1.

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the zeta subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of Mr 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTzeta-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.