Alternative Splicing of NOX4 in the Failing Human Heart

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5'-3' end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure

Diamond-based sensors for in vitro cellular radiobiology: Simultaneous detection of cell exocytic activity and ionizing radiation

The investigation of secondary effects induced by ionizing radiation represents a new and ever-growing research field in radiobiology. This new paradigm cannot be investigated only using standard instrumentation and methodologies, but rather requires novel technologies to achieve significant progress. In this framework, we developed diamond-based sensors that allow simultaneous real-time measurements with a high spatial resolution of the secretory activity of a network of cells cultured on the device, as well as of the dose at which they are exposed during irradiation experiments. The devices were functionally characterized by testing both the above-mentioned detection schemes, namely: amperometric measurements of neurotransmitter release from excitable cells (such as dopamine or adrenaline) and dosimetric evaluation using different ionizing particles (alpha particle and X-ray photons). Finally, the sensors were employed to investigate the effects induced by X-rays on the exocytotic activity of PC12 neuroendocrine cells by monitoring the modulation of the dopamine release in real-time

Results from CHIPIX-FE0, a Small Scale Prototype of a New Generation Pixel Readout ASIC in 65nm CMOS for HL-LHC

CHIPIX65-FE0 is a readout ASIC in CMOS 65nm designed by the CHIPIX65 project for a pixel detector at the HL-LHC, consisting of a matrix of 64x64 pixels of dimension 50x50 μm2. It is fully functional, can work at low thresholds down to 250e− and satisfies all the specifications. Results confirm low-noise, fast performance of both the synchronous and asynchronous front-end in a complex digital chip. CHIPIX65-FE0 has been irradiated up to 600 Mrad and is only marginally affected on analog performance. Further irradiation to 1 Grad will be performed. Bump bonding to silicon sensors is now on going and detailed measurements will be presented. The HL-LHC accelerator will constitute a new frontier for particle physics after year 2024. One major experimental challenge resides in the inner tracking detectors, measuring particle position: here the dimension of the sensitive area (pixel) has to be scaled down with respect to LHC detectors. This paper describes the results obtained by CHIPIX65-FE0, a readout ASIC in CMOS 65nm designed by the CHIPIX65 project as small-scale demonstrator for a pixel detector at the HL-LHC. It consists of a matrix of 64x64 pixels of dimension 50x50 um2 pixels and contains several pieces that are included in RD53A, a large scale ASIC designed by the RD53 Collaboration: two out of three front-ends (a synchronous and an asynchronous architecture); several building blocks; a (4x4) pixel region digital architecture with central local buffer storage, complying with a 3 GHz/cm2 hit rate and a 1 MHz trigger rate maintaining a very high efficiency (above 99%). The chip is 100% functional, either running in triggered or trigger-less mode. All building-blocks (DAC, ADC, Band Gap, SER, sLVS-TX/RX) and very front ends are working as expected. Analog performance shows a remarkably low ENC of 90e-, a fast-rise time below 25ns and low-power consumption (about 4μA/pixel) in both synchronous and asynchronous front-ends; a very linear behavior of CSA and discriminator. No significant cross talk from digital electronics has been measured, achieving a low threshold of 250e-. Signal digitization is obtained with a 5b-Time over Threshold technique and is shown to be fairly linear, working well either at 80 MHz or with higher frequencies of 300 MHz obtained with a tunable local oscillator. Irradiation results up to 600 Mrad at low temperature (-20°C) show that the chip is still fully functional and analog performance is only marginally degraded. Further irradiation will be performed up to 1 Grad either at low or room temperature, to further understand the level of radiation hardness of CHIPIX65-FE0. We are now in the process of bump bonding CHIPIX65-FE0 to 3D and possibly planar silicon sensors during spring. Detailed results will be presented in the conference paper

First Measurements of a Prototype of a New Generation Pixel Readout ASIC in 65 nm CMOS for Extreme Rate HEP Detectors at HL-LHC

A first prototype of a readout ASIC in CMOS 65nm for a pixel detector at High Luminosity LHC is described. The pixel cell area is 50x50 um2 and the matrix consists of 64x64 pixels. The chip was designed to guarantee high efficiency at extreme data rates for very low signals and with low power consumption. Two different analogue front-end designs, one synchronous and one asynchronous, were implemented, both occupying an area of 35x35 um2. ENC value is below 100e- for an input capacitance of 50 fF and in-time threshold below 1000e-. Leakage current compensation up to 50 nA with power consumption below 5 uW. A ToT technique is used to perform charge digitization with 5-bit precision using either a 40 MHz clock or a local Fast Oscillator up to 800 MHz. Internal 10-bit DAC's are used for biasing, while monitoring is provided by a 12-bit ADC. A novel digital architecture has been developed to ensure above 99.5% hit efficiency at pixel hit rates up to 3 GHz/cm2, trigger rates up to 1 MHz and trigger latency of 12.5 us. The total power consumption per pixel is below 5uW. Analogue dead-time is below 1%. Data are sent via a serializer connected to a CMOS-to-SLVS transmitter working at 320 MHz. All IP-blocks and front-ends used are silicon-proven and tested after exposure to ionizing radiation levels of 500-800 Mrad. The chip was designed as part of the Italian INFN CHIPIX65 project and in close synergy with the international CERN RD53 and was submitted in July 2016 for production. Early test results for both front-ends regarding minimum threshold, auto-zeroing and low-noise performance are high encouraging and will be presented in this paper

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche’s Endocannabinoid System Projects

: The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions