NADPH as a potential intrinsic probe for tumour margin estimation

The fluorescent properties of the reduced coenzyme NADH and its phosphorylated derivative (NADPH) have been explored in order to assess their potential as an intrinsic probe for cancer surgery. NADPH production is increased in cancer cells to quench reactive oxygen species and meet higher demands for biosynthesis, and has attractive fluorescent properties such as emission towards the visible part of the spectrum and a relatively long fluorescence lifetime upon binding to enzymes (~ 1 – 6.5 ns) that helps discriminate against other endogenous species. Different environmental effects on NAD(P)H fluorescence are reported here, including an increase in lifetime upon oxygen removal, an ability to retain its fluorescent properties in a complex medium (a silica phantom) and its fluorescence lifetime also being distinguishable in a cell environment. In addition, the development of a miniaturized liquid light guide filter-based timecorrelated single photon counting fluorescence lifetime system is reported as a step towards time-resolved visual imaging in cancer surgery. This system has been demonstrated as being capable of accurately measuring NAD(P)H fluorescence lifetimes in both simple solvent and cellular environments

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

Fluorescence guided surgery

Fluorescence guided surgery (FGS) is an imaging technique that allows the surgeon to visualise different structures and types of tissue during a surgical procedure that may not be as visible under white light conditions. Due to the many potential advantages of fluorescence guided surgery compared to more traditional clinical imaging techniques such as its higher contrast and sensitivity, less subjective use, and ease of instrument operation, the research interest in fluorescence guided surgery continues to grow over various key aspects such as fluorescent probe development and surgical system development as well as its potential clinical applications. This review looks to summarise some of the emerging opportunities and developments that have already been made in fluorescence guided surgery in recent years while highlighting its advantages as well as limitations that need to be overcome in order to utilise the full potential of fluorescence within the surgical environment

Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain-of-function toxicity mechanism contributing to the pathogenesis of fragile X-associated premature ovarian insufficiency

Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by expansion of a CGG trinucleotide repeat sequence located in the 5’ untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. Women with FXPOI have a depleted ovarian follicle population, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by expansions of the CGG sequence to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or a protein-based mechanism, where repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. Here, we have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that are specifically sequestered by CGG RNA aggregates in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Furthermore, we have demonstrated reduced expression of three proteins identified via our RNA pulldown (FUS, PA2G4 and TRA2β) in ovarian follicles in a FMR1 premutation mouse model. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology

A robust approach to differentiate human monocyte-derived microglia from peripheral blood mononuclear cells

Microglia are implicated in most neurodegenerative diseases. Here, we present a robust and efficient protocol to differentiate monocyte-derived microglia-like cells (MDMi) from whole blood. The protocol consists of three parts. The first part will describe two methods for PBMC isolation. This will be followed by MDMi differentiation, and lastly, the characterization of MDMi by immunocytochemistry. MDMi can be used to investigate microglial-related responses in various age-related neurodegenerative diseases and can be applied to drug testing on a personalized basis. For complete details on the use and execution of this protocol, please refer to Quek et al

Nanoparticle metrology of silica colloids and super-resolution studies using the ADOTA fluorophore

We describe how a new fluorescent dye, methyl ADOTA (N-methyl-azadioxatriangulenium tetrafluoroborate), is an improvement on dyes reported previously for measuring silica nanoparticle size in sols using the decay of fluorescence anisotropy. Me(thyl)-ADOTA possesses the unusual combination of having a red emission and a long fluorescence lifetime of ~ 20 ns, leaving it better-placed to reveal particle sizes at the upper end of the 1-10 nm measurement range. For stable LUDOX colloids, Me-ADOTA is shown to offer higher measurement precision in ≤ 1/30th of the measurement time required for dyes previously used. In measurement times of only ~ 20 mins nanoparticle radii for LUDOX SM-AS, AM and AS-40 of 4.6 ± 0.3 nm, 5.9 ± 0.2 nm and 11.1 ± 1.1 nm, are in good agreement with two of the manufacturer’s values of 3.5 nm, 6 nm and 11 nm respectively. Unlike the Si-ADOTA (N-(4-(triethoxysilylethyl)urea-phenyl-) ADOTA tetrafluoroborate) derivative containing a reactive trimetoxysilane group, Me-ADOTA is shown to not induce aggregation of colloidal silica. Measurements on nanoparticles growing in an acidic silica hydrogel at pH 0.94, prior to the gel time of ~ 50 hr, reveals an average nanoparticle size up to ~ 6.3 nm, significantly larger than the 4.5 nm reported previously. The difference is most certainly due to the longer fluorescence lifetime of Me-ADOTA (~ 20 ns) revealing the presence of larger particles. Studies of growing silica clusters in an alcogel of tetraethyl orthosilicate (TEOS) were able to resolve a monotonically increasing average radius of 1.42 ± 0.10 nm to 1.81 ± 0.14 nm over a period of 48 hr. We have also assessed a carboxylic acid derivative of ADOTA (N-(3-carboxypropylene)-ADOTA tetrafluoroborate - Acid-ADOTA) using dSTORM super-resolution microscopy. Although demonstrating high photochemical stability and blinking, its lower brightness and relative propensity to aggregate limits Acid-ADOTA’s use for dSTORM

Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system