4FISH-IF,a Four-Color Dual-Gene FISH Combined with p63 Immunofluorescence to Evaluate NKX3.1 and MYC Status in Prostate Cancer

NKX3.1 allelic loss and MYC amplification are common events during prostate cancer progression and have been recognized as potential prognostic factors in prostate cancer after radical prostatectomy or precision radiotherapy. We have developed a 4FISH-IF assay (a dual-gene fluorescence in situ hybridization combined with immunofluorescence) to measure both NKX3.1 and MYC status on the same slide. The 4FISH-IF assay contains four probes complementary to chromosome 8 centromere, 8p telomere, 8p21, and 8q24, as well as an antibody targeting the basal cell marker p63 visualized by immunofluorescence. The major advantages of the 4FISH-IF include the distinction between benign and malignant glands directly on the 4FISH-IF slide and the control of truncation artifact. Importantly, this specialized and innovative combined multiprobe and immunofluorescence technique can be performed on diagnostic biopsy specimens, increasing its clinical relevance. Moreover, the assay can be easily performed in a standard clinical molecular pathology laboratory. Globally, the use of 4FISH-IF decreases analytic time, increases confidence in obtained results, and maintains the tissue morphology of the diagnostic specimen.     

TLR3, TLR4 and TLRs7–9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.     

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen, or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.     

Recovery of vegetation in a Caledonian pinewood after fire

Summary

Following a wildfire in the Caledonian pinewood at Shieldaig, Wester Ross in late March 1974, the recovery of the vegetation, particularly Scots pine (Pinus sylvestris) seedlings, was monitored in twenty sample plots. Almost all trees under 5 cm diameter at breast height were immediately killed by the fire and of the larger trees, 45 per cent of the Scots pine and 60 per cent of the birch (Betula pendula) had died by 1980. Regeneration of Scots pine had occurred to the extent of about 2,500 seedlings ha^-1 by 1980. Recovery of the field layer species varied; species with rhizomes grew up quickly but the re-establishment of cover by Calluna vulgaris and many bryophytes was slow.     

High-throughput,multi-platform metabolomics on very small volumes: 1H NMR metabolite identification in an unadulterated tube-in-tube system

As small animal models of disease become more widely used, there is increasing importance and potential for characterizing their metabolomes. However, as the animal becomes smaller, the amounts of biofluids such as urine and cerebral spinal fluid available for metabolomic studies are more limited. Further, in multi-platform systems biology when the same small sample must be used for several analyses, it is a frequent requirement that no additions are made to the sample (even as simple as D₂O or an NMR chemical shift reference) to maintain sample integrity. Herein we describe a method for high-throughput ¹H-NMR studies using ~30 µl volumes, suitable for biofluid matrices. The compartmentalization of the sample and NMR standards, however, requires chemical shift corrections due to bulk magnetic susceptibility and ionic strength changes for metabolite profiling using a reference library or data-binning of the chemical shift axis. This set-up minimizes the cost of individual data collection per small animal and is suitable for high-throughput, longitudinal, multimodal metabolomic studies of biofluids available in limited quantities.     

Cephalopods in neuroscience: regulations,research and the 3Rs

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of “live cephalopods” became regulated within the European Union by Directive 2010/63/EU on the “Protection of Animals used for Scientific Purposes”, giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce “guidelines” and the potential contribution of neuroscience research to cephalopod welfare.     

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.