Plasma membrane potential oscillations in insulin secreting Ins-1 832/13 cells do not require glycolysis and are not initiated by fluctuations in mitochondrial bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.     

Novel phantom for performance evaluation of contrast-enhanced 3D rotational angiography

PurposeThis technical note presents an in-house phantom with a specially designed contrast-object module constructed to address the need for three-dimensional rotational angiography (3DRA) testing.MethodsThe initial part of the study was a brief evaluation on the commercially available phantom used for 3DRA and computed tomography angiography (CTA) to confirm the need for a special phantom for 3D angiography. Once confirmed, an in-house phantom was constructed. The novel phantom was tested to evaluate the basic image performance metrics, i.e., unsharpness (MTF) and noise characterization (NPS), as well as to show its capability for vessel contrast visibility study.ResultsThe low contrast objects in the commercially available tools dedicated for CT is found to yield significantly lower signal difference to noise ratio (SDNR) when used for 3DRA, therefore deemed inadequate for 3DRA contrast evaluation. The constructed in-house phantom demonstrates a capability to serve for basic imaging performance check (MTF, NPS, and low contrast evaluation) for 3DRA and CTA. With higher and potentially adjustable visibility of contrast objects as artificial vessels, the in-house phantom also makes more clinically relevant tests, e.g., human- or model observer study and task-based optimization, possible.ConclusionThe novel phantom with special contrast object module shows higher visibility in 3DRA compared to the currently available commercial phantom and, therefore, is recommended for use in 3D angiography.     

Mass spectrometry techniques for imaging and detection of metallodrugs

Undoubtedly, metallomic approaches based on mass spectrometry have evolved into essential tools supporting the drug development of novel metal-based anticancer drugs. This article will comment on the state-of-the-art instrumentation and highlight some of the recent analytical advances beyond routine, especially focusing on the latest developments in inductively coupled plasma-mass spectrometry (ICP-MS). Mass spectrometry-based bioimaging and single-cell methods will be presented, paving the way to exciting investigations of metal-based anticancer drugs in heterogeneous and structurally, as well as functionally complex solid tumor tissues.     

Unstructured loop is essential for the activation of mGluR2

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or “BC loop”) in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.     

Single-crystal structure and intracellular localization of Zn(II)-thiosemicarbazone complex targeting mitochondrial apoptosis pathways

Tracking of drugs in cancer cells is important for basic biology research and therapeutic applications. Therefore, we designed and synthesised a Zn(II)-thiosemicarbazone complex with photoluminescent property for organelle-specific imaging and anti-cancer proliferation. The Zn(AP44eT)(NO₃)₂ coordination ratio of metal to ligand was 1:1, which was remarkably superior to 2-((3-aminopyridin-2-yl) methylene)-N, N-diethylhydrazinecarbothioamide (AP44eT·HCl) in many aspects, such as fluorescence and anti-tumour activity. Confocal fluorescence imaging showed that the Zn(AP44eT)(NO₃)₂ was aggregated in mitochondria. Moreover, Zn(AP44eT)(NO₃)₂ was more effective than the metal-free AP44eT·HCl in shortening the G2 phase in the MCF-7 cell cycle and promoting apoptosis of cancer cells. Supposedly, the effects of these complexes might be located mainly in the mitochondria and activated caspase-3 and 9 proteins.     

Combination of dual-energy computed tomography and iterative metal artefact reduction to increase general quality of imaging for radiotherapy patients with high dense materials. Phantom study

PurposeTo evaluate the use of pseudo-monoenergetic reconstructions (PMR) from dual-energy computed tomography, combined with the iterative metal artefact reduction (iMAR) method.MethodsPseudo-monoenergetic CT images were obtained using the dual-energy mode on the Siemens Somatom Definition AS scanner. A range of PMR combinations (70–130 keV) were used with and without iMAR. A Virtual Water™ phantom was used for quantitative assessment of error in the presence of high density materials: titanium, alloys 330 and 600. The absolute values of CT number differences (AD) and normalised standard deviations (NSD) were calculated for different phantom positions. Image quality was assessed using an anthropomorphic pelvic phantom with an embedded hip prosthesis. Image quality was scored blindly by five observers.ResultsAD and NSD values revealed differences in CT number errors between tested sets. AD and NSD were reduced in the vicinity of metal for images with iMAR (p < 0.001 for AD/NSD). For ROIs away from metal, with and without iMAR, 70 keV PMR and pCT AD values were lower than for the other reconstructions (p = 0.039). Similarly, iMAR NSD values measured away from metal were lower for 130 keV and 70 keV PMR (p = 0.002). Image quality scores were higher for 70 keV and 130 keV PMR with iMAR (p = 0.034).ConclusionThe use of 70 keV PMR with iMAR allows for significant metal artefact reduction and low CT number errors observed in the vicinity of dense materials. It is therefore an attractive alternative to high keV imaging when imaging patients with metallic implants, especially in the context of radiotherapy planning.     

Accuracy of predicting chemical body composition of growing pigs using dual-energy X-ray absorptiometry

Studies in animal science assessing nutrient and energy efficiency or determining nutrient requirements benefit from gathering exact measurements of body composition or body nutrient contents. Those are acquired by standardized dissection or by grinding the body followed by wet chemical analysis, respectively. The two methods do not result in the same type of information, but both are destructive. Harnessing human medical imaging techniques for animal science can enable repeated measurements of individuals over time and reduce the number of individuals required for research. Among imaging techniques, dual-energy X-ray absorptiometry (DXA) is particularly promising. However, the measurements obtained with DXA do not perfectly match dissections or chemical analyses, requiring the adjustment of the DXA via calibration equations. Several calibration regressions have been published, but comparative studies of those regression equations and whether they are applicable to different data sets are pending. Thus, it is currently not clear whether existing regression equations can be directly used to convert DXA measurements into chemical values or whether each individual DXA device will require its own calibration. Our study builds prediction equations that relate body composition to the content of single nutrients in growing entire male pigs (BW range 20–100 kg) as determined by both DXA and chemical analyses, with R² ranging between 0.89 for ash and 0.99 for water and CP. Moreover, we show that the chemical composition of the empty body can be satisfactorily determined by DXA scans of carcasses, with the prediction error ranging between 4.3% for CP and 12.6% for ash. Finally, we compare existing prediction equations for pigs of a similar range of BWs with the equations derived from our DXA measurements and evaluate their fit with our chemical analysis data. We found that existing equations for absolute contents that were built using the same DXA beam technology predicted our data more precisely than equations based on different technologies and percentages of fat and lean mass. This indicates that the creation of generic regression equations that yield reliable estimates of body composition in pigs of different growth stages, sexes and genetic breeds could be achievable in the near future. DXA may be a promising tool for high-throughput phenotyping for genetic studies, because it efficiently measures body composition in a large number and wide array of animals.     

使用非侵入的神经影像方法解码人类思维

随着神经影像技术的发展,“读脑机器”在不久的将来可能会变成现实。在众多实现“读脑机器”的技术中,使用基于血氧水平依赖的脑功能磁共振技术的神经解码方法则是非常有希望的一种技术。在本报告中,我们结合我们实验室最近开展的两个研究工作,说明了使用功能磁共振技术进行神经解码的一般方法及其应用。本领域的发展很快,所有的这些工作让我们对发明“读脑机器”充满了期待。但是,与此同时,我们也应该看到目前还存在着很多技术的局限和挑战。本报告的最后,我们也对这些局限和挑战进行了一些讨论。     

The multicompartmental p32/gClqR as a new target for antibody-based tumor targeting strategies

Tumor-associated cell surface antigens and tumor-associated vascular markers have been used as a target for cancer intervention strategies. However, both types of targets have limitations due to accessibility, low and/or heterogeneous expression, and presence of tumor-associated serum antigen. It has been previously reported that a mitochondrial/cell surface protein, p32/gC1qR, is the receptor for a tumor-homing peptide, LyP-1, which specifically recognizes an epitope in tumor cells, tumor lymphatics, and tumor-associated macrophages/myeloid cells. Using antibody phage technology, we have generated an anti-p32 human monoclonal antibody (2.15). The 2.15 antibody, expressed in single-chain fragment variable and in trimerbody format, was then characterized in vivo using mice grafted subcutaneously with MDA-MB-231 human breast cancers cells, revealing a highly selective tumor uptake. The intratumoral distribution of the antibody was consistent with the expression pattern of p32 in the surface of some clusters of cells. These results demonstrate the potential of p32 for antibody-based tumor targeting strategies and the utility of the 2.15 antibody as targeting moiety for the selective delivery of imaging and therapeutic agents to tumors.