Elucidating Regulatory Mechanisms of Cardiac CaV1.2 and NaV1.5 Channels

In the heart, sodium (Na+) influx via NaV1.5 channels initiates the action potential, and calcium (Ca2+) influx via CaV1.2 channels has a key role in excitation-contraction coupling and determining the plateau phase of the action potential. Mutations in the genes that encode these ion channels or in proteins that modulate them are linked to arrhythmias and cardiomyopathy, underscoring the need for characterizing mechanisms of regulation. The work presented in this thesis is subdivided into three different chapters, each with a distinct focus on ion channel modulation. The first chapter details our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Physiologic β-adrenergic activation of PKA during the sympathetic “fight or flight” response increases Ca2+ influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. Recent data suggest that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. To test if any non-conserved sites are required for regulation, we generated mice with inducible cardiac-specific expression of α1C with mutations at both conserved and non- conserved predicted PKA phosphorylation sites (35-mutant α1C). Additionally, we createdanother mouse with inducible cardiac-specific expression of β2 with mutations at predicted PKA phosphorylation sites (28-mutant β2B). In each of these mice, β-adrenergic stimulation of Ca²⁺ current was unperturbed. Finally, to test the hypothesis that redundant functional PKA phosphorylation sites exist on the α1C subunit and β2 subunit or that several sites confer incremental regulation, we crossed the 35-mutant α1C mice with the 28-mutant β2B mice to generate offspring expressing both mutant subunits. In these offspring, intact regulation was observed. These results provide the definitive answer that phosphorylation of the α1C subunit or β2 subunit is not required for β-adrenergic regulation of CaV1.2 in the heart. In the second chapter, we study the influence of calmodulin and fibroblast growth homologous factor (FHF) FGF13 on late Na+ current. Studies in heterologous expression systems show that the Ca²⁺-binding protein calmodulin plays a key role in decreasing late Na⁺ current. The effect of loss of calmodulin binding to NaV1.5 on late Na+ current has yet to be resolved in native cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA), disrupting calmodulin binding to the C-terminus. Surprisingly, we found that the IQ/AA mutation did not cause an increase late Na⁺ current in cardiomyocytes. These findings suggest the existence of endogenous protective mechanisms that counteract the increase in late Na+ current that occurs with loss of calmodulin binding. We reasoned that FGF13, a known modulator of late Na+ current that is endogenously expressed in cardiomyocytes but not HEK cells, might play a protective role in limiting late Na+ current. Finally, we coexpressed the IQ/AA mutant NaV1.5 channel in HEK293 cells with FGF13 and found that FGF13 diminished the late Na⁺ currentcompared to cells without FGF13, suggesting that endogenous FHFs may serve to prevent late Na⁺ current in mouse cardiomyocytes. The third chapter of this thesis focuses on the use of proximity labeling and multiplexed quantitative proteomics to define changes in the NaV1.5 macromolecular complex in Duchenne muscular dystrophy (DMD), in which the absence of dystrophin predisposes affected individuals to arrhythmias and cardiac dysfunction.. Standard methods to characterize macromolecular complexes have relied on candidate immunoprecipitation or immunocytochemistry techniques that fall short of providing a comprehensive view of the numbers and types of interactors, as well as the potential dynamic nature of the interactions that may be perturbed by disease states. To provide an inclusive understanding of NaV1.5 macromolecular complexes, we utilize live-cell APEX2 proximity labeling in cardiomyocytes. We identify several proximal changes that align with the electrophysiological NaV1.5 phenotype of young dystrophin-deficient mice, including a decrease in Ptpn3 and Gdp1l and an increase in proteasomal machinery. Whole-cell protein expression fold-change results were used to reveal the altered global expression profile and to place context behind NaV1.5-proximal changes. Finally, we leveraged the neighborhood- specificity of proteins at the lateral membrane, intercalated disc, and transverse tubules of cardiomyocytes to demonstrate that NaV1.5 channels can traffic to all three membrane compartments even in the absence of dystrophin. Thus, the approach of proximity labeling in cardiomyocytes from an animal model of human disease offers new insights into molecular mechanisms of NaV1.5 dysfunction in DMD and provides a template for similar investigations in other cardiac diseases

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the prometastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis

The SNS linac high power RF system design, status, and results

The Spallation Neutron Source being built at the Oak Ridge National Lab in Tennessee requires a 1 GeV proton linac. Los Alamos has responsibility for the RF systems for the entire linac. The linac requires 3 distinct types of RF systems: 2.5-MW peak, 402.5 MHz, RF systems for the RFQ and DTL (7 systems total); 5-MW peak, 805 MHz systems for the CCL and the two energy corrector cavities (6 systems total); and 550-kW peak, 805 MHz systems for the superconducting sections (8 1 systems total). The design of the SNS Linac RF system was presented at the 2001 Particle Accelerator Conference in Chicago. Vendors have been selected for the klystrons (3 different vendors), circulators ( I vendor), transmitter (1 vendor), and high power RF loads (3 different vendors). This paper presents the results and status of vendor procurements, test results of the major components of the Linac RF system and our installation progress

Diminished cell proliferation promotes natural killer cell adaptive-like phenotype by limiting FcεRIγ expression.

Human adaptive-like natural killer (NK) cells express low levels of FcεRIγ (FcRγ-/low) and are reported to accumulate during COVID-19 infection; however, the mechanism underlying and regulating FcRγ expression in NK cells has yet to be fully defined. We observed lower FcRγ protein expression in NK cell subsets from lung transplant patients during rapamycin treatment, suggesting a link with reduced mTOR activity. Further, FcRγ-/low NK cell subsets from healthy donors displayed reduced mTOR activity. We discovered that FcRγ upregulation is dependent on cell proliferation progression mediated by IL-2, IL-15, or IL-12, is sensitive to mTOR suppression, and is inhibited by TGFβ or IFNα. Accordingly, the accumulation of adaptive-like FcRγ-/low NK cells in COVID-19 patients corresponded to increased TGFβ and IFNα levels and disease severity. Our results show that an adaptive-like NK cell phenotype is induced by diminished cell proliferation and has an early prognostic value for increased TGFβ and IFNα levels in COVID-19 infection associated with disease severity

Diminished cell proliferation promotes natural killer cell adaptive-like phenotype by limiting FcεRIγ expression.

Human adaptive-like natural killer (NK) cells express low levels of FcεRIγ (FcRγ-/low) and are reported to accumulate during COVID-19 infection; however, the mechanism underlying and regulating FcRγ expression in NK cells has yet to be fully defined. We observed lower FcRγ protein expression in NK cell subsets from lung transplant patients during rapamycin treatment, suggesting a link with reduced mTOR activity. Further, FcRγ-/low NK cell subsets from healthy donors displayed reduced mTOR activity. We discovered that FcRγ upregulation is dependent on cell proliferation progression mediated by IL-2, IL-15, or IL-12, is sensitive to mTOR suppression, and is inhibited by TGFβ or IFNα. Accordingly, the accumulation of adaptive-like FcRγ-/low NK cells in COVID-19 patients corresponded to increased TGFβ and IFNα levels and disease severity. Our results show that an adaptive-like NK cell phenotype is induced by diminished cell proliferation and has an early prognostic value for increased TGFβ and IFNα levels in COVID-19 infection associated with disease severity

Merkel Cells Activate Sensory Neural Pathways through Adrenergic Synapses

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of β2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch

convertibleCARs: A chimeric antigen receptor system for flexible control of activity and antigen targeting.

We have developed a chimeric antigen receptor (CAR) platform that functions as a modular system to address limitations of traditional CAR therapies. An inert form of the human NKG2D extracellular domain (iNKG2D) was engineered as the ectodomain of the CAR to generate convertibleCARTM-T cells. These cells were specifically directed to kill antigen-expressing target cells only in the presence of an activating bispecific adapter comprised of an iNKG2D-exclusive ULBP2-based ligand fused to an antigen-targeting antibody (MicAbodyTM). Efficacy against Raji tumors in NSG mice was dependent upon doses of both a rituximab-based MicAbody and convertibleCAR-T cells. We have also demonstrated that the exclusive ligand-receptor partnering enabled the targeted delivery of a mutant form of IL-2 to selectively promote the expansion of convertibleCAR-T cells in vitro and in vivo. By altering the Fv domains of the MicAbody or the payload fused to the orthogonal ligand, convertibleCAR-T cells can be readily targeted or regulated

Silicon Solar Cells with Front Hetero-Contact and Aluminum Alloy Back Junction Preprint SILICON SOLAR CELLS WITH FRONT HETERO-CONTACT AND ALUMINUM ALLOY BACK JUNCTION

ABSTRACT We prototype an alternative n-type monocrystalline silicon (c-Si) solar cell structure that utilizes an n/i-type hydrogenated amorphous silicon (a-Si:H) front heterocontact and a back p-n junction formed by alloying aluminum (Al) with the n-type Si wafer. Such a structure combines a conventional high-throughput Al-Si alloying process with excellent front surface passivation provided by a-Si:H. A key process consideration is to preserve the clean c-Si front surface through the high-temperature alloying, so there will be effective a-Si:H passivation. From cell simulations, we estimate a front SRV of 10-50 cm/sec has been achieved in our process. The best prototype 1×1 cm 2 cell with planar front surface and single anti-reflection (AR) coating layer has demonstrated a confirmed conversion efficiency of 13.5%, Voc of 604.7 mV, and fill factor (FF) of 79.9%. Processes for further efficiency improvements are described

Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo.

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression for the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Our data and workflow thus provide a uniquely coherent in vivo lncRNA analysis and form the foundation of a user-friendly online resource for the study of lncRNAs in development and disease

Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo.

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression for the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Our data and workflow thus provide a uniquely coherent in vivo lncRNA analysis and form the foundation of a user-friendly online resource for the study of lncRNAs in development and disease