The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: Identification of the nuclear pore complex as a potential target of the JNK pathway

c-Jun is induced in many neuronal death paradigms. A critical step in c-Jun regulation involves phosphorylation of Ser63/Ser73 located in the NH2-terminal transactivation domain. To determine the importance of this phosphorylation for neuronal apoptosis, we analyzed the sympathetic neurons of mice carrying a mutant c-Jun gene that lacks Ser63/Ser73 phosphorylation sites (jun aa). Trophic factor–deprivation or DNA damage–induced death was significantly delayed in jun aa/aa neurons. Neuronal c-Jun induction was only partially inhibited, demonstrating that phosphorylation of Ser63/73 is not required for c-Jun activation. The inductions of proapoptotic BH3-only proteins, Bim and PUMA/Bbc3, were delayed during neuronal apoptosis in mutant neurons. These results demonstrate that NH2-terminal c-Jun phosphorylation is important, but not necessary, for the induction of proapoptotic genes and neuronal apoptosis. Thus, additional JNK substrates may be critical for neuronal death. As potential mediators, we identified additional nuclear MLK/JNK substrates, including Nup214 subunit of the nuclear pore complex

A 0.4 nJ Excitation Energy Bridge-to-Digital Converter for Implantable Pulmonary Artery Pressure Monitoring

This paper presents an energy-efficient, duty-cycled, and spinning excitation bridge-to-digital converter (BDC) suitable for measuring the pulmonary artery pressure of heart failure patients with an implantable system. The duty-cycled bridge uses resistances of 6.2 kΩ and, with a supply of 1.2 V, consumes 0.4 nJ excitation energy. A novel spinning method is applied to the bridge and the capacitive DAC simultaneously in such a way to achieve an offset-independent digital output and to eliminate the need for complex instrumentation amplifiers with offset-reduction techniques or calibration. The SAR ADC fabricated in 0.18-μm CMOS consumes 19 nW at 1.2 V. With a sampling rate of 1 kS/s, the converter achieves the ENOB of 9.2 bits

Smartphone-based, rapid, wide-field fundus photography for diagnosis of pediatric retinal diseases

PurposeAn important, unmet clinical need is for cost-effective, reliable, easy-to-use, and portable retinal photography to evaluate preventable causes of vision loss in children. This study presents the feasibility of a novel smartphone-based retinal imaging device tailored to imaging the pediatric fundus.MethodsSeveral modifications for children were made to our previous device, including a child-friendly 3D printed housing of animals, attention-grabbing targets, enhanced image stitching, and video-recording capabilities. Retinal photographs were obtained in children undergoing routine dilated eye examination. Experienced masked retina-specialist graders determined photograph quality and made diagnoses based on the images, which were compared to the treating clinician's diagnosis.ResultsDilated fundus photographs were acquired in 43 patients with a mean age of 6.7 years. The diagnoses included retinoblastoma, Coats' disease, commotio retinae, and optic nerve hypoplasia, among others. Mean time to acquire five standard photographs totaling 90-degree field of vision was 2.3 ± 1.1 minutes. Patients rated their experience of image acquisition favorably, with a Likert score of 4.6 ± 0.8 out of 5. There was 96% agreement between image-based diagnosis and the treating clinician's diagnosis.ConclusionsWe report a handheld smartphone-based device with modifications tailored for wide-field fundus photography in pediatric patients that can rapidly acquire fundus photos while being well-tolerated.Translational relevanceAdvances in handheld smartphone-based fundus photography devices decrease the technical barrier for image acquisition in children and may potentially increase access to ophthalmic care in communities with limited resources

Improved limit on the directly measured antiproton lifetime

Continuous monitoring of a cloud of antiprotons stored in a Penning trap for 405 days enables us to set an improved limit on the directly measured antiproton lifetime. From our measurements we extract a storage time of 3.15x108 equivalent antiproton-seconds, resulting in a lower lifetime limit of Tp > 10.2,a with a confidence level of 68%. This result improves the limit on charge-parity-time violation in antiproton decays based on direct observation by a factor of 7

Sixfold improved single particle measurement of the magnetic moment of the antiproton

Our current understanding of the Universe comes, among others, from particle physics and cosmology. In particle physics an almost perfect symmetry between matter and antimatter exists. On cosmological scales, however, a striking matter/antimatter imbalance is observed. This contradiction inspires comparisons of the fundamental properties of particles and antiparticles with high precision. Here we report on a measurement of the g-factor of the antiproton with a fractional precision of 0.8 parts per million at 95% confidence level. Our value /2=2.7928465(23) outperforms the previous best measurement by a factor of 6. The result is consistent with our proton g-factor measurement gp/2=2.792847350(9), and therefore agrees with the fundamental charge, parity, time (CPT) invariance of the Standard Model of particle physics. Additionally, our result improves coefficients of the standard model extension which discusses the sensitivity of experiments with respect to CPT violation by up to a factor of 20.EU/ERC/290870-MEFUCOMax-Planck SocietyHelmholtz-GemeinschaftRIKEN Initiative Research Unit ProgramRIKEN President FundingRIKEN Pioneering Project FundingRIKEN FPR FundingRIKEN JRA ProgramMEXT/24000008Max-Planck SocietyEU/ERC Advanced Grant/290870-MEFUCOHelmholtz-GemeinschaftCERN-fellowship program

Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity

Protein kinase D (PKD) is a novel family of serine/threonine kinases regulated by diacylglycerol, which is involved in multiple cellular processes and various pathological conditions. The limited number of cell-active, selective inhibitors has historically restricted biochemical and pharmacological studies of PKD. We now markedly expand the PKD1 inhibitory chemotype inventory with eleven additional novel small molecule PKD1 inhibitors derived from our high throughput screening campaigns. The in vitro IC50s for these eleven compounds ranged in potency from 0.4 to 6.1 µM with all of the evaluated compounds being competitive with ATP. Three of the inhibitors (CID 1893668, (1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one; CID 2011756, 5-(3-chlorophenyl)-N-[4-(morpholin-4-ylmethyl)phenyl]furan-2-carboxamide; CID 5389142, (6Z)-6-[4-(3-aminopropylamino)-6-methyl-1H-pyrimidin-2-ylidene]cyclohexa-2,4-dien-1-one) inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. The specificity of these compounds for PKD1 inhibitory activity was supported by kinase assay counter screens as well as by bioinformatics searches. Moreover, computational analyses of these novel cell-active PKD1 inhibitors indicated that they were structurally distinct from the previously described cell-active PKD1 inhibitors while computational docking of the new cell-active compounds in a highly conserved ATP-binding cleft suggests opportunities for structural modification. In summary, we have discovered novel PKD1 inhibitors with in vitro and cell-based inhibitory activity, thus successfully expanding the structural diversity of small molecule inhibitors available for this important pharmacological target

CDK-Mediated Regulation of Cell Functions via c-Jun Phosphorylation and AP-1 Activation

Cyclin-dependent kinases (CDKs) and their targets have been primarily associated with regulation of cell-cycle progression. Here we identify c-Jun, a transcription factor involved in the regulation of a broad spectrum of cellular functions, as a newly recognized CDK substrate. Using immune cells from mouse and human, and several complementary in vitro and in vivo approaches including dominant negative protein expression, pharmacologic inhibitors, kinase assays and CDK4 deficient cells, we demonstrate the ability of CDK4 to phosphorylate c-Jun. Additionally, the activity of AP-1, a ubiquitous transcription factor containing phosphorylated c-Jun as a subunit, was inhibited by abrogating CDK4. Surprisingly, the regulation of c-Jun phosphorylation by CDK4 occurred in non-dividing cells, indicating that this pathway is utilized for cell functions that are independent of proliferation. Our studies identify a new substrate for CDK4 and suggest a mechanism by which CDKs can regulate multiple cellular activation functions, not all of which are directly associated with cell cycle progression. These findings point to additional roles of CDKs in cell signaling and reveal potential implications for therapeutic manipulations of this kinase pathway

Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner

Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death

Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF