Coleywalton9762
The introduction of fluorescent detection systems has revolutionized the applicability of Western blotting for quantitative protein expression analyses. The fundamental premise behind fluorescent Western blotting is the combination of distinct fluorescent dye-conjugated secondary antibodies and high performance digital imaging solutions in which the fluorescence signal is directly proportional to the amount of protein enabling quantitative measurements and simultaneous detection of several target proteins. This aspect of Western blotting is now widely used, especially in preclinical research, to detect quantitative changes in protein levels and phosphorylation status between experimental groups. This chapter provides a detailed step-by-step guide for best practice procedures during the entire process from sample preparation, SDS polyacrylamide gel electrophoresis to electrotransfer of proteins and highlights approaches that can be applied to increase data output.Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method. However, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry. We recently developed a method that applies CyTOF (Cytometry by Time-Of-Flight mass spectrometry) to high-dimensional analysis of individual human synaptosomes, overcoming many of the multiplexing limitations of conventional flow cytometry. We call this new method Mass Synaptometry. Here we describe the preparation of synaptosomes from human and mouse brain, the generation and quality control of the "SynTOF" (Synapse by Time-Of-Flight mass spectrometry) antibody panel, the staining protocol, and CyTOF parameter setup for acquisition, post-acquisition processing, and analysis.For many years real-time quantitative polymerase chain reaction (qPCR) has been the golden standard to measure gene expression levels in brain tissue. However, today it is generally accepted that many factors may affect the outcome of the study and more consensus is required to perform and interpret real-time qPCR experiments in a comparable way. Here we describe the basic techniques used for more than a decade in our laboratory to extract RNA and protein from the same piece of frozen brain tissue and to quantify relative mRNA levels with real-time qPCR and SYBR Green.Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H+-ATPases. Since H+ gradient across SV membrane drives neurotransmitter uptake into SVs, precise measurements of steady-state vesicular pH and dynamics of re-acidification process will provide important information concerning the H+-driven neurotransmitter uptake. Indeed, we recently demonstrated distinct features of steady state and dynamics of vesicular pH between glutamatergic vesicles and GABAergic vesicles in cultured hippocampal neurons. In this article, we focus on an experimental protocol and setup required to determine steady-state luminal pH of SVs in living neurons. This protocol is composed of efficient expression of a pH-sensitive fluorescent protein in the lumen of SVs in cultured neurons, and recordings of its fluorescence changes under a conventional fluorescent microscope during local applications of acidic buffer and ionophores-containing solution at a given pH. The method described here can be easily applied for measuring luminal pH of different types of secretory organelles and other acidic organelles such as lysosomes and endosomes in cultured cell preparations.The analysis of organellar membrane transporters presents many technical problems. In general, their activity depends on a H+ electrochemical driving force (ΔμH+). However, transport itself influences the expression of ΔμH+ in standard radiotracer flux assays, making it difficult to disentangle the role of the chemical component ΔpH and the membrane potential Δψ. Whole endosome recording in voltage clamp circumvents many of these problems, controlling ionic conditions as well as membrane potential inside and outside the organelle . This approach has been used primarily to study the properties of endolysosomal channels, which generate substantial currents (Saito et al., J Biol Chem 282(37)27327-27333, 2007; Cang et al., Nat Chem Biol 10(6)463-469, 2014; Cang et al., Cell 152(4)778-790, 2013; Chen et al., Nat Protoc 12(8)1639-1658, 2017; Samie et al., Dev Cell 26(5)511-524, 2013; Wang et al., Cell 151(2)372-383, 2012). Electrogenic transport produces much smaller currents, but we have recently reported the detection of transport currents and an uncoupled Cl- conductance associated with the vesicular glutamate transporters (VGLUTs) that fill synaptic vesicles with glutamate (Chang et al., eLife 7e34896, 2018). In this protocol, we will focus on the measurement of transport currents on enlarged endosomes of heterologous mammalian cells.Live-imaging of axonal cargoes within central nervous system has been a long-lasting interest for neurobiologists as axonal transport plays critical roles in neuronal growth, function, and survival. Many kinds of cargoes are transported within axons, including synaptic vesicles and a variety of membrane-bound and membrane-less organelles. Imaging these cargoes at high spatial and temporal resolution, and within living brains, is technically very challenging. Here, we describe a quantitative method, based on customized mounting chambers, allowing live-imaging of axonal cargoes transported within the maturing brain of the fruit fly, Drosophila melanogaster. With this method, we could visualize in real time, using confocal microscopy, cargoes transported along axons. Our protocol is simple and easy to set up, as brains are mounted in our imaging chambers and ready to be imaged in about 1 h. Another advantage of our method is that it can be combined with pharmacological treatments or super-resolution microscopy.Neuronal miRNAs play major roles in regulation of synaptic development and plasticity. The small size of miRNAs and, in some cases, their low level of expression make their quantification and detection challenging. Here, we outline methods to quantify steady state levels of miRNAs in neurons and the brain by using real-time quantitative PCR (RT-qPCR) and to determine miRNA subcellular localization in primary neurons by a sensitive fluorescence in situ hybridization (FISH) method.
Urine is conventionally used as a specimen to document diazepam-related crimes; however, few reports have described the pharmacokinetics of diazepam and its metabolites in urine.
This study aimed to investigate the pharmacokinetics of diazepam and its metabolites, including glucuronide compounds, in the urine of Chinese participants.
A total of 28 volunteers were recruited and each participant ingested 5 mg of diazepam orally. Ten milliliters of urine were collected from each participant at post-consumption timepoints of prior (zero), 1, 2, 4, 8, 12, and 24 h and 2, 3, 6, 12, and 15 days. All samples were extracted by solid-phase extraction and analyzed using high-performance liquid chromatography-tandem mass spectrometry. Diazepam and its main metabolites, except for temazepam, were detected in the urine of volunteers. Pharmacokinetic parameters were analyzed using the pharmacokinetic software DAS according to the non-compartment model.
Urinary diazepam peaked at 2.38 ng/mL (C
) and 1.93 h (T
). The urinary metabolite nordiazepam peaked at 1.17 ng/mL and 100.21 h; temazepam glucuronide (TG) peaked at 145.61 ng/mL and 41.14 h; and oxazepam glucuronide (OG) peaked at 101.57 ng/mL and 165.86 h. The elimination half-life (t
) and clearance (CLz/F) for diazepam were 119.58 h and 65.77 L/h, respectively. The t
of the metabolites nordiazepam, TG, and OG was 310.58 h, 200.17 h, and 536.44 h, respectively. Finally, this study found that both diazepam and its main metabolites in urine were detectable for at least 15 days, although there were individual differences.
The results regarding diazepam pharmacokinetics in urine would be of great help in forensic science and drug screening.
The results regarding diazepam pharmacokinetics in urine would be of great help in forensic science and drug screening.
The European Risk Management Plan (EU-RMP) is a proactive planning tool for identification, characterisation and management of important risks and missing information throughout the lifecycle of a medicinal product. Over the past 15 years the EU-RMP has been a part of the pharmacovigilance practice in Europe, but there are no published studies assessing impact of the growing experience and evolving regulatory framework on the content and focus of the EU-RMP.
The objectives were to study the real-world impact of evolving pharmacovigilance guidelines on the proactive lifecycle management of important risks and missing information through EU-RMPs, and to further explore the impact of different resources on the management of the benefit-risk profile.
A retrospective study based on the review of 64 EU-RMPs dated between 01 January 2006 and 01 October 2020 for seven human medicinal products for which Boehringer Ingelheim holds the Marketing Authorisation in the European Union. Data on the timing and rational for keeping an up-to-date understanding of a medicinal product's safety profile. The aim of improving the efficiency of risk management has leveraged the accumulation of knowledge leading to revision of regulatory guidelines and increasingly, proactive Risk Management Plans focused on safety concerns that are important for patients and public health.
Automated insulin delivery (AID) systems can enable improved glycaemic outcomes with reduced mental burden. Open-source AID (OS-AID) systems overcome some of the developmental and access barriers enabling a wider use of these systems. selleck Limited data are available on healthcare professional (HCP) opinions and current practice regarding these systems. The aim of this survey was to gain insight into HCP perceptions and practices around OS-AID.
This survey was developed collaboratively with OS-AID users and distributed to adult and children's teams, using an online survey tool. Results were received between February and April 2019. Responses were assessed using simple descriptive statistics with analyses stratified by respondent characteristics.
317 responses were obtained from a range of HCPs in both adult and paediatric services. Key results include HCP perception of OS-AID as "risky in the wrong hands" (43%); 91% felt uncomfortable initiating discussions around OS-AID because of lack of regulation (67%) and/or their own lack of knowledge (63%). Half of HCPs (47%) reported that they would choose OS-AID if they themselves had type 1 diabetes.
HCPs are generally supportive of OS-AID users but many feel uncomfortable with the technicalities of the systems given the lack of approval. Knowledge around the use of these systems was limited. Re-assessment of HCP perceptions should be performed in the future given the evolving landscape of diabetes technology, recent consensus statements and emerging ethical and legal perspectives.
HCPs are generally supportive of OS-AID users but many feel uncomfortable with the technicalities of the systems given the lack of approval. Knowledge around the use of these systems was limited. Re-assessment of HCP perceptions should be performed in the future given the evolving landscape of diabetes technology, recent consensus statements and emerging ethical and legal perspectives.
