Fast fluorescence dynamics in non-ratiometric calcium indicators

A fluorescence decay of high-affinity non-ratiometric Ca2+ indicator Oregon Green BAPTA-1 (OGB-1) is analyzed with unprecedented temporal resolution in the two-photon excitation regime. A triple exponential decay is shown to best fit the fluorescence dynamics of OGB-1. We provide a new model for accurate measurements of the free Ca2+ concentration and dissociation constants of non-ratiometric calcium indicators.Comment: 3 pages, 2 figures, figures revised, added chi-square goodness of fi

Ultra Compact and Low-power TDC and TAC Architectures for Highly-Parallel Implementation in Time-Resolved Image Sensors

We report on the design and characterization of three different architectures, namely two Time-to- Digital Converters (TDCs) and a Time-to-Amplitude Converter (TAC) with embedded analog-to-digital conversion, implemented in a 130-nm CMOS imaging technology. The proposed circuit solutions are conceived for implementation at pixel-level, in image sensors exploiting Single-Photon Avalanche Diodes as photodetectors. The fabricated 32x32 TDCs/TACs arrays have a pitch of 50μm in both directions while the average power consumption is between 28mW and 300mW depending on the architectural choice. The TAC achieves a time resolution of 160ps on a 20-ns time range with a differential and integral non-linearity (DNL, INL) of 0.7LSB and 1.9LSB, respectively. The two TDCs have a 10-bit resolution with a minimum time resolution between 50ps and 119ps and a worst-case accuracy of ±0.5 LSB DNL and 2.4 LSB INL. An overview of the performance is given together with the analysis of the pros and cons for each architecture

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mousemodel of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force.This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.Muscular Dystrophy Association (Award MDA277360)National Institutes of Health (U.S.) (Grant 5DP1-MH100706)National Institutes of Health (U.S.) (Grant R01DK097768

A 32x32 50ps Resolution 10 bit Time to Digital Converter Array in 130nm CMOS for Time Correlated Imaging

We report the design and characterisation of a 32x32 time to digital (TDC) converter plus single photon avalanche diode (SPAD) pixel array implemented in a 130nm imaging process. Based on a gated ring oscillator approach, the 10 bit, 50mm pitch TDC array exhibits a minimum time resolution of 50ps, with accuracy of ±0.5 LSB DNL and 4 LSB INL. Process, voltage and temperature compensation (PVT) is achieved by locking the array to a stable external clock. The resulting time correlated pixel array is a viable candidate for single photon counting (TCSPC) applications such as fluorescent lifetime imaging microscopy (FLIM), nuclear or 3D imaging and permits scaling to larger array formats

PARP1 is required for adhesion molecule expression in atherogenesis

Aims Atherosclerosis is the leading cause of death in Western societies and a chronic inflammatory disease. However, the key mediators linking recruitment of inflammatory cells to atherogenesis remain poorly defined. Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme, which plays a role in acute inflammatory diseases. Methods and results In order to test the role of PARP in atherogenesis, we applied chronic pharmacological PARP inhibition or genetic PARP1 deletion in atherosclerosis-prone apolipoprotein E-deficient mice and measured plaque formation, adhesion molecules, and features of plaque vulnerability. After 12 weeks of high-cholesterol diet, plaque formation in male apolipoprotein E-deficient mice was decreased by chronic inhibition of enzymatic PARP activity or genetic deletion of PARP1 by 46 or 51%, respectively (P < 0.05, n ≥ 9). PARP inhibition or PARP1 deletion reduced PARP activity and diminished expression of inducible nitric oxide synthase, vascular cell adhesion molecule-1, and P- and E-selectin. Furthermore, chronic PARP inhibition reduced plaque macrophage (CD68) and T-cell infiltration (CD3), increased fibrous cap thickness, and decreased necrotic core size and cell death (P < 0.05, n ≥ 6). Conclusion Our data provide pharmacological and genetic evidence that endogenous PARP1 is required for atherogenesis in vivo by increasing adhesion molecules with endothelial activation, enhancing inflammation, and inducing features of plaque vulnerability. Thus, inhibition of PARP1 may represent a promising therapeutic target in atherosclerosi

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR/Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR/Cas9-based genome editing as a potential therapy to treat DMD