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Skeletal muscle integrates many of the systemic signals, which contribute to the adaptive remodeling, and beneficial effects of exercise. One mechanism through which muscle mediates the systemic effects of exercise is through muscle-derived hormones known as myokines. We identified the metabolite β-aminoisobutyric acid (BAIBA) as an exercise-mediated small molecule myokine. BAIBA is secreted from muscle in response to increased expression of the transcriptional co-regulator PGC-1α, a master regulator of the muscle adaptive response to exercise. However, how BAIBA functions to regulate the adaptive responses of skeletal muscle to exercise remains poorly understood. We show that BAIBA improves muscle metabolism, exercise efficiency and performance in mice. Oxygen consumption (VO2) and energy expenditure are increased in BAIBA-treated mice. Furthermore, BAIBA increased soleus in situ muscle contractile force, fatigue resistance, mitochondrial number and function. We found that BAIBA drives muscle fiber-type switching to an oxidative phenotype in vivo. BAIBA regulates specific fiber-type gene expression in human myocytes through PPARΔ. Our findings demonstrate that BAIBA is a key paracrine myokine, which, in part, regulates the effects of exercise to improve muscle function with resultant effects on exercise performance. H.N.D. acknowledges the support of the Biotechnology and Biological Sciences Research Council (BB/T004231/1). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Cascadia subduction zone (CSZ) is an exceptional geologic environment for recording evidence of land-level changes, tsunamis, and ground motion that reveals at least 19 great megathrust earthquakes over the past 10 kyr. Such earthquakes are among the most impactful natural hazards on Earth, transcend national boundaries, and can have global impact.Reducing the societal impacts of future events in the US Pacific Northwest and coastal British Columbia, Canada, requires improved scientific understanding of megathrust earthquake rupture, recurrence, and corresponding hazards. Despite substantial knowledge gained from decades of research, large uncertainties remain about the characteristics and frequencies of past CSZ earthquakes. In this review, we summarize geological, geophysical, and instrumental evidence relevant to understanding megathrust earthquakes along the CSZ and associated uncertainties. We discuss how the evidence constrains various models of great megathrust earthquake recurrence in Cascadia and identify potential paths forward for the earthquake science community. ▪ Despite outstanding geologic records of past megathrust events, large uncertainty of the magnitude and frequency of CSZ earthquakes remains. ▪ This review outlines current knowledge and promising future directions to address outstanding questions on CSZ rupture characteristics and recurrence. ▪ Integration of diverse data sets with attention to the geologic processes that create different records has potential to lead to major progress.

Studies of recent destructive megathrust earthquakes and tsunamis along subduction margins in Japan, Sumatra, and Chile have linked forearc morphology and structure to megathrust behavior. This connection is based on the idea that spatial variations in the frictional behavior of the megathrust influence the tectono-morphological evolution of the upper plate. Here we present a comprehensive examination of the tectonic geomorphology, outer wedge taper, and structural vergence along the marine forearc of the Cascadia subduction zone (offshore northwestern North America). The goal is to better understand geologic controls on outer wedge strength and segmentation at spatial scales equivalent to rupture lengths of large earthquakes (≥M 6.7), and to examine potential linkages with shallow megathrust behavior.We use cross-margin profiles, spaced 25 km apart, to characterize along-strike variation in outer wedge width, steepness, and structural vergence (measured between the toe and the outer arc high). The width of the outer wedge varies between 17 and 93 km, and the steepness ranges from 0.9° to 6.5°. Hierarchical cluster analysis of outer wedge width and steepness reveals four distinct regions that also display unique patterns of structural vergence and shape of the wedge: Vancouver Island, British Columbia, Canada (average width, linear wedge, seaward and mixed vergence); Washington, USA (higher width, concave wedge, landward and mixed vergence); northern and central Oregon, USA (average width, linear and convex wedge, mixed and seaward vergence); and southern Oregon and northern California, USA (lower width, convex wedge, seaward and mixed vergence). Variability in outer wedge morphology and structure is broadly associated with along-strike megathrust segmentation inferred from differences in oceanic asthenospheric velocities, patterns of episodic tremor and slow slip, GPS models of plate locking, and the distribution of seismicity near the plate interface. In more detail, our results appear to delineate the extent, geometry, and lithology of dynamic and static backstops along the margin. Varying backstop configurations along the Cascadia margin are interpreted to represent material-strength contrasts within the wedge that appear to regulate the along- and across-strike taper and structural vergence in the outer wedge. We argue that the morphotectonic variability in the outer wedge may reflect spatial variations in shallow megathrust behavior occurring over roughly the last few million years. Comparing outer wedge taper along the Cascadia margin to a global compilation suggests that observations in the global catalog are not accurately representing the range of heterogeneity within individual margins and highlights the need for detailed margin-wide morphotectonic analyses of subduction zones worldwide.

Reduction of matched and nearly complementary unintended transcripts was evaluated for 96 antisense oligonucleotides (ASOs) and 832 nearly matched unintended transcripts. The ASOs were 16–20 nucleotide “gapmers” with a gap of 8–10 DNA residues and 2′-O-methoxy-ethyl or constrained-ethyl substitutions in the wings. Most unintended transcripts were not reduced or were reduced with a potency more than 10-fold weaker than the intended transcript. For the unintended transcripts that were reduced, a strong correlation between relative potency of the intended versus the unintended transcript with predicted free energy of hybridization was observed. These results suggest ASO selectivity should be evaluated by testing for reduction of the unintended transcripts predicted to bind most stably to the ASO.

β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.

Coding variants (named G1 and G2) in Apolipoprotein L1 (APOL1) can explain most excess risk of kidney disease observed in African American individuals. It has been proposed that risk variant APOL1 dose, such as increased risk variant APOL1 level serves as a trigger (second hit) for disease development. The goal of this study was to determine whether lowering risk variant APOL1 levels protects from disease development in a podocyte-specific transgenic mouse disease model. We administered antisense oligonucleotides (ASO) targeting APOL1 to podocyte-specific G2APOL1 mice and observed efficient reduction of APOL1 levels. APOL1 ASO1, which more efficiently lowered APOL1 transcript levels, protected mice from albuminuria, glomerulosclerosis, tubulointerstitial fibrosis, and renal failure. Administration of APOL1 ASO1 was effective even for established disease in the NEFTA-rtTA/TRE-G2APOL1 (NEFTA/G2APOL1) mice. We observed a strong correlation between APOL1 transcript level and disease severity. We concluded that APOL1 ASO1 may be an effective therapeutic approach for APOL1-associated glomerular disease.

The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 hours. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to subfertility.

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 h. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2 h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to infertility.

SummaryThere are at least two distinct types of thermogenic adipocyte in mammals: a pre-existing form established during development, termed classical brown adipocytes and an inducible form, ‘beige’ adipocytes1–3. Various environmental cues can stimulate a process frequently referred to as ‘beiging’ of white adipose tissue (WAT), leading to enhanced thermogenesis and obesity resistance 4, 5. Whilst beiging of WAT as a therapeutic goal for obesity and obesity-related complications has attracted much attention6–9; therapeutics stimulating beiging without deleterious side-effects remain elusive10. The endothelium lines all blood vessels and is therefore in close proximity to all cells. Many studies support the possibility that the endothelium acts as a paracrine organ11–14. We explored the potential role of endothelial insulin-like growth factor-1 receptor (IGF-1R) as a paracrine modulator of WAT phenotype. Here we show that a reduction in endothelial IGF-1R expression in the presence of nutrient excess leads to white adipocyte beiging, increases whole-body energy expenditure and enhances insulin sensitivity via a non-cell autonomous paracrine mechanism. We demonstrate that this is mediated by endothelial release of malonic acid, which we show, using prodrug analogues, has potentially therapeutically-relevant properties in the treatment of metabolic disease.

Changes in composition of the intestinal microbiota are linked to the development of obesity and can lead to endothelial cell (EC) dysfunction. It is unknown whether EC can directly influence the microbiota. Insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) are critical for coupling nutritional status and cellular growth; IGF-1R is expressed in multiple cell types including EC. The role of ECIGF-1R in the response to nutritional obesity is unexplored. To examine this, we use gene-modified mice with EC-specific overexpression of human IGF-1R (hIGFREO) and their wild-type littermates. After high-fat feeding, hIGFREO weigh less, have reduced adiposity and have improved glucose tolerance. hIGFREO show an altered gene expression and altered microbial diversity in the gut, including a relative increase in the beneficial genus Akkermansia. The depletion of gut microbiota with broad-spectrum antibiotics induces a loss of the favourable metabolic differences seen in hIGFREO mice. We show that IGF-1R facilitates crosstalk between the EC and the gut wall; this crosstalk protects against diet-induced obesity, as a result of an altered gut microbiota.

Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole-body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endothelial growth factor A (VEGF-A), including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial nitric oxide synthase was intact, but downstream signaling to extracellular signal-reduced kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2. ispartof: ENDOCRINOLOGY vol:162 issue:8 ispartof: location:United States status: published

The endoplasmic reticulum (ER) regulates cellular protein and lipid biosynthesis. ER dysfunction leads to protein misfolding and the unfolded protein response (UPR), which limits protein synthesis to prevent cytotoxicity. Chronic ER stress in skeletal muscle is a unifying mechanism linking lipotoxicity to metabolic disease. Unidentified signals from cells undergoing ER stress propagate paracrine and systemic UPR activation. Here, we induce ER stress and lipotoxicity in myotubes. We observe ER stress-inducing lipid cell non-autonomous signal(s). Lipidomics identifies that palmitate-induced cell stress induces long-chain ceramide 40:1 and 42:1 secretion. Ceramide synthesis through the ceramide synthase 2 de novo pathway is regulated by UPR kinase Perk. Inactivation of CerS2 in mice reduces systemic and muscle ceramide signals and muscle UPR activation. The ceramides are packaged into extracellular vesicles, secreted and induce UPR activation in naïve myotubes through dihydroceramide accumulation. This study furthers our understanding of ER stress by identifying UPR-inducing cell non-autonomous signals.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE−/−) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE−/− and Nox2 (ESMIRO/ApoE−/−/Nox2−/y) were generated and compared with ESMIRO/ApoE−/−/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE−/− mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE−/−/Nox2−/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE−/−/Nox2−/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE−/− mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

The California Seafloor and Coastal Mapping Program (CSCMP) is a collaborative effort to develop comprehensive bathymetric, geologic, and habitat maps and data for California's State Waters. CSCMP began in 2007 when the California Ocean Protection Council (OPC) and the National Oceanic and Atmospheric Administration (NOAA) allocated funding for high-resolution bathymetric mapping, largely to support the California Marine Life Protection Act and to update nautical charts. Collaboration and support from the U.S. Geological Survey and other partners has led to development and dissemination of one of the world's largest seafloor-mapping datasets. CSCMP provides essential science and data for ocean and coastal management, stimulates and enables research, and raises public education and awareness of coastal and ocean issues. Specific applications include: • Delineation and designation of marine protected areas • Characterization and modeling of benthic habitats and ecosystems • Updating nautical charts • Earthquake hazard assessments • Tsunami hazard assessments • Planning offshore infrastructure • Providing baselines for monitoring change • Input to models of sediment transport, coastal erosion, and coastal flooding • Regional sediment management • Understanding coastal aquifers • Providing geospatial data for emergency response

Introduction Insulin resistance is an independent risk factor for cardiovascular disease. We have previously shown that the insulin-like growth factor-1 receptor (IGF-1R) is a negative regulator of insulin receptor (IR) signalling, by sequestering IR subunits in insulin resistant IR:IGF1R ‘hybrid receptors’. By crossing IR haploinsufficient (IRKO) mice with IGF-1R haploinsufficient (IGF-1RKO) mice (producing “double knockout” or DKO mice), we have previously demonstrated rescue of the endothelial dysfunction caused by insulin resistance. Whether this is associated with wider benefits in vascular biology is unclear. Our original hypothesis is that reduced expression of the IGF-1R in whole body insulin resistance improves vascular repair and regeneration. Methods Metabolic assessment included measurement of weight gain and glucose and insulin tolerance testing. Denuding femoral artery endothelial injury was induced with angioplasty guidewire and repair was quantified by Evans Blue perfusion 4 days later. For hindlimb ischemia, the left femoral artery was ligated and excised, with sham surgery contralaterally. Control:ischemic limb perfusion ratio was assessed with weekly laser Doppler imaging for 4 weeks. Data are expressed as mean (standard error) and compared using t-tests; * denotes p Results Glucose and insulin tolerance tests were similar in DKO and IRKO mice. Body weight was significantly lower in DKO than IRKO [area under curve (arbitrary units) 116(2.1) Vs 123(2.4)* n=15]. DKO had a significantly greater proportion of recovered endothelium (Figure 1 A&B) after denuding wire injury to the femoral artery compared with IRKO [0.55 (0.04) Vs 0.46 (0.02) p=0.047*, n=8-14]. ]. DKO had superior recovery (Figure 2 A&B) after induction of hindlimb ischemia [area under curve limb perfusion ratio (arbitrary units) 2.2(0.11) Vs 1.3(0.08)* n=12-19]. Conclusion Reduced IGF-1R expression improves vascular repair and regeneration in the context of whole-body insulin resistance. Further work will aim to elucidate the possible mechanisms for these novel observations. Conflict of interest None

Reduced IGF-1 signalling is an evolutionarily conserved mediator of longevity, yet the magnitude of this effect is substantially larger in organisms retaining a common insulin and IGF-1 receptor. Whether this discrepancy reflects the failure to simultaneously reduce IGF-1 and insulin signalling in mammalian model systems remains unexplored. Moreover, studies of invertebrates cannot ascertain whether substantial effects upon lifespan are associated with preserved cognitive performance, a crucial component of healthspan. We compared the healthspan of male mice with haploinsufficiency of the insulin receptor (IRKO), IGF-1 receptor (IGF-1RKO), or both (DKO), with wildtype (WT) littermates. DKO mice survived longer than WT, with IRKO and IGF-1RKO being intermediate. At 2 years of age, DKO also exhibited preserved nesting behaviour in contrast with all other genotypes. Differential insulin sensitivity or weight gain during ageing did not explain the preserved healthspan of DKO, since these were comparable to IRKO littermates. These data provide the first demonstration that reduced insulin and IGF-1 signalling have synergistic effects upon healthspan in a mammalian model system, suggesting future mechanistic and translational studies should target insulin and IGF-1 signalling.

Systemic lupus erythematosus is an autoimmune disease that manifests in widespread complement activation and deposition of complement fragments in the kidney. The complement pathway is believed to play a significant role in the pathogenesis and in the development of lupus nephritis. Complement factor B is an important activator of the alternative complement pathway and increasing evidence supports reducing factor B as a potential novel therapy to lupus nephritis. Here we investigated whether pharmacological reduction of factor B expression using antisense oligonucleotides could be an effective approach for the treatment of lupus nephritis. We identified potent and well tolerated factor B antisense oligonucleotides that resulted in significant reductions in hepatic and plasma factor B levels when administered to normal mice. To test the effects of factor B antisense oligonucleotides on lupus nephritis, we used two different mouse models, NZB/W F1 and MRL/lpr mice, that exhibit lupus nephritis like renal pathology. Antisense oligonucleotides mediated reductions in circulating factor B levels were associated with significant improvements in renal pathology, reduced glomerular C3 deposition and proteinuria, and improved survival. These data support the strategy of using factor B antisense oligonucleotides for treatment of lupus nephritis in humans.

An addressable array test structure is proposed for characterization of transistor variability beyond 3σ away from the mean. The design of the array is based on very compact basic cells which enable a highly efficient layout which has over three times higher normalized device density than similar arrays. Implementations of a 32k array are demonstrated for placement in a standard wafer scribe lane module. Characterization results based on an advanced high-k/metal gate process show that transistor threshold voltages follow a Gaussian distribution at current levels typically used in digital circuits. Analysis of random and systematic components of variability confirms that there are no systematic spatial gradients across the array and that random variations account for 99% of total variability.

Statistical analysis plan. (PDF 114 kb)

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We use >10,000 km of high-resolution seismic-reflection data together with multibeam bathymetry to document complex and highly variable post-Last Glacial Maximum (LGM) sediment distribution and thickness in the coastal zone (~10 m isobath to 5.6 km offshore) along a ~800 km section of central California's transform continental margin. Sediment thickness ranges from 0 (seafloor bedrock) to 64 m with a mean of 8.7 m. We delineate 25 coastal zone “sediment domains,” and group them based on common geomorphology and sediment occurrence. Thickest sediment occurs in “mountain front” and “large river” domains, which comprise 14.5% and 7.9% of the coastal zone and contain 30.1% and 18.2% of coastal zone sediment, respectively. In contrast, “small river” domains and “sediment-poor shelf” domains comprise 50.7% and 15.7% of the coastal zone and contain 18.4% and 12.7% of its sediment. The distribution and thickness of post-LGM sediment in the coastal zone is controlled by a combination of tectonics, sediment supply, and eustasy. Sediment is derived from a tectonically controlled coastal landscape of rapidly uplifting mountain fronts, more slowly uplifting marine terraces, and fault-bounded headlands and alluvial-estuarine troughs. Sediment supply is maximized along steep, landslide-prone, mountain fronts and at the mouths of large watersheds, and minimized along lower-relief, terraced coastal landscape drained by smaller rivers and creeks. In the offshore coastal zone, tectonics generates local uplifts and basins, and influences shelf width and gradient as well as the locations of some shelf-incised submarine canyons. Sea-level rise raises base level, drowns estuaries, creates accommodation space on the shelf (amount based on gradient), and isolates the heads of many submarine canyons at or near the shelfbreak. Comparison of shelf sediment volumes with estimates of “unaltered” watershed sediment supply reveals that a relatively small proportion of post-LGM sediment supply is preserved on the shelf offshore of some of the largest rivers. Sediments deposited in shoreline and shelf environments have limited preservation potential, and the most complete long-term geologic record of the post-LGM transgression and highstand is likely represented in slope and submarine fan deposits.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis. The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a cryptic 3' splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern. We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype. Moreover, we compared the effect of 2'-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2'-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia. In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence. Disclosures Dong: Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths in the world. Disease subtypes include lung adenocarcinoma (LUAD), which accounts for nearly 50% of lung malignancies, and lung squamous carcinoma (LUSC). Disease diagnosis at late stages and limited therapeutic options are the major causes for the poor prognosis of lung cancer patients. Additionally, lack of discriminatory diagnostic and prognostic biomarkers have hampered therapeutic management and, consequently, NSCLC patients are treated as one entity. In this modern era, the development of next generation based RNA sequencing (RNA-Seq) methodologies enable us to accurately quantify the transcriptional abundance in cells in a highly reproducible and reliable manner. By utilizing this technology, we sequenced a large cohort of lung cancer samples and leveraged several publically available RNA-Seq datasets for analysis to facilitate a deeper understanding of the disease. Furthermore, from the large RNA-Seq datasets, we also discovered long noncoding RNAs (lncRNA). These transcripts, which are longer than 200 nucleotides that are not translated into protein, have tissue-, lineage-, and cancer-specific expression patterns. In our previous study, we developed a highly significant prognostic marker gene signature by analyzing 255 LUAD patients from The Cancer Genome Atlas (TCGA) dataset. Using a training cohort, 96 genes including 5 lncRNAs had significant prognostic associations with lung cancer. Further, a stepwise regression model identified a four-gene signature, including one lncRNA (LADDER). The four-gene signature was then validated in two independent patient cohorts from the TCGA and in-house data. Functional studies showed that inhibition of LADDER lncRNA using both siRNA and ASOs decreased cell proliferation in lung cancer cells. In addition, knockdown of LADDER lncRNA using ASOs decreased lung cancer cell growth in vivo. Taken together, we have identified the lncRNA LADDER, a member of a four-gene prognostic signature, to play a functional role in lung cancer progression as well as serve as a robust prognostic marker. Note: This abstract was not presented at the meeting. Citation Format: Sathiya Pandi Narayanan, Sudhanshu Shukla, Jean Tien, Palak Shah, Sunita Shankar, Sethuramasundaram Pitchaiya, Srihari Srinivasan, Yuping Zhang, Xiaoming Wang, Lanbo Xiao, Xuhong Cao, Susan Freier, Andrew Watt, Shuling Guo, Felix Feng, David Beer, Saravana M. Dhanasekaran, Arul M. Chinnaiyan. Identification and functional characterization of prognostic long non coding RNA LADDER in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3573.

Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target. The Treg lineage is defined by expression of the FOXP3 transcription factor, which controls immune-suppressive functions. We have developed the clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform). AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR. Tregs treated with AZD8701 further exhibited reduced suppressive functions in in vitro suppression assays, which confirmed the functional effects of FOXP3 modulation. Finally, AZD8701 promoted dose-dependent knockdown of FOXP3 in humanized mice, including >50% FOXP3 knockdown at doses that can be feasibly achieved with the Gen 2.5 ASO platform in the clinic. To support the importance of FOXP3 in immuno-oncological settings, we characterized murine surrogate FOXP3 ASOs in the context of syngeneic tumour bearing mice. Murine FOXP3 ASOs similarly promoted >50% FOXP3 knockdown in mice and were well tolerated with no overt toxicological findings at high doses, over a maximum of 12 weeks of treatment. Murine FOXP3 ASOs significantly attenuated tumour growth in A20 and ID8-VEGF syngeneic models, which was associated with some complete tumour regressions. Moreover, we found that mouse surrogate FOXP3 ASOs promoted additive/enhanced therapeutic effects when combined with immune checkpoint blockade. Collectively, FOXP3 ASOs represent a first-in-class strategy to target Tregs in cancer in a highly selective manner. The clinical application of AZD8701 may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blocking agents. Citation Format: Charles Sinclair, Alexey S. Revenko, R B. Johnson, Alison Peter, Molly A. Taylor, Lisa A. Hettrick, Stephanie Klein, Anisha Solanki, Melissa Chapman, James Yates, Helen K. Angell, Andrew Watt, Danielle Gattis, Brett P. Monia, Simon T. Barry, Paul Lyne, Mark Edbrooke, Frederick Goldberg, A R. Macleod. Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2713.

Rev-Erb-α and Rev-Erb-β are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)-HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5' ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Zinc, the most abundant trace metal in the brain, has numerous functions in health and disease. It is released into the synaptic cleft alongside glutamate and this connection between zinc and glutamatergic neurotransmission allows the ion to modulate overall excitability of the brain and influence synaptic plasticity. To maintain healthy synapses, extracellular zinc levels need to be tightly regulated. We recently reported that the cellular prion protein (PrP (C) ) can directly influence neuronal zinc concentrations by promoting zinc uptake via AMPA receptors. The octapeptide repeat region of PrP (C) is involved in zinc sensing or scavenging and the AMPA receptor provides the channel for transport of the metal across the membrane, facilitated by a direct interaction between the N-terminal polybasic region of PrP (C) and AMPA receptors. PrP (C) has been evolutionarily linked to the Zrt/Irt-like protein (ZIP) metal ion transport family with the C-terminus of PrP (C) sharing sequence similarities with the N-terminal extracellular domains of ZIP 5, 6 and 10. By incorporating the properties of ZIP transporters (both zinc sensing and zinc transport) into two existing neuronal proteins, (PrP (C) as zinc sensor, AMPA receptor as zinc transporter), neuronal cells are enhancing their biological efficiency and functionality.

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.