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The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors. The Rpd3L HDAC complex is an ancient chromatin-modifying complex found in diverse eukaryotes. Here, authors describe the cryo-EM structure of the yeast complex and show that key features are preserved in the human complex.

Transverse aortic constriction (TAC) is a well-established model of pressure overload-induced cardiac hypertrophy and failure in mice. The degree of constriction “tightness” dictates the TAC severity and is determined by the gauge (G) of needle used. Though many reports use the TAC model, few studies have directly compared the range of resulting phenotypes. In this study adult male mice were randomized to receive TAC surgery with varying degrees of tightness: mild (25G), moderate (26G) or severe (27G) for 4 weeks, alongside sham-operated controls. Weekly echocardiography and terminal haemodynamic measurements determined cardiac remodelling and function. All TAC models induced significant, severity-dependent left ventricular hypertrophy and diastolic dysfunction compared to sham mice. Mice subjected to 26G TAC additionally exhibited mild systolic dysfunction and cardiac fibrosis, whereas mice in the 27G TAC group had more severe systolic and diastolic dysfunction, severe cardiac fibrosis, and were more likely to display features of heart failure, such as elevated plasma BNP. We also observed renal atrophy in 27G TAC mice, in the absence of renal structural, functional or gene expression changes. 25G, 26G and 27G TAC produced different responses in terms of cardiac structure and function. These distinct phenotypes may be useful in different preclinical settings.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We developed a novel “two-hit” model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57Bl6/NJ mice fed a high-fat diet (HFD) for > 10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d . HFD-Renin (aka “HFpEF”) mice demonstrated obesity and insulin resistance, moderate left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by echocardiographic measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to human HFpEF. Treatment with empagliflozin, an effective but incompletely understood HFpEF therapy, improved multiple endpoints. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Additional preclinical HFpEF models allow for orthogonal studies to increase validity in assessment of interventions.

cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation after pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules that mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload and in regulating BP. In a transaortic constriction HF model, PKG activation with sildenafil preserved LV function in MLK3+/+ but not MLK3-/- littermates. MLK3 coimmunoprecipitated with PKG1α. MLK3-PKG1α cointeraction decreased in failing LVs. PKG1α phosphorylated MLK3 on Thr277/Ser281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the soluble guanylate cyclase (sGC) stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserved LV function, whereas MLK3 kinase-independent signaling regulated BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.

It is unknown how the timing between doses might affect nicotine's impact on neural activity. Our objective was to examine how the interdose interval affects nicotine's impact on resting-state functional connectivity (rsFC). Adult male Sprague-Dawley rats were administered nicotine daily (0.4 mg/kg) over 6 days while control animals received saline vehicle. Functional magnetic resonance imaging was used to measure rsFC before and after a challenge dose of nicotine (0.4 mg/kg) delivered for the first time and 3, 6, 12, or 24hr after the previous dose. As the interval between nicotine doses increased from 3 to 24hr, the strength of rsFC increased in some circuits, particularly the nucleus accumbens and prefrontal circuits, and decreased in others, namely the interpeduncular nucleus, hippocampus, caudoputamen, retrosplenial cortex, ventral tegmental, and the insular circuits. These data indicate that the effect that nicotine has on the brain is affected by the amount of time that has passed since the previous dose. The effect on rsFC of cumulative doses is not additive. This may have important implications for the study of nicotine addiction as it implies that the same dose of nicotine might have a different impact on the brain depending on the time elapsed from the previous exposure.

Individuals with attention deficit/hyperactivity disorder (ADHD) are susceptible to earlier and more severe nicotine addiction. To shed light on the relationship between nicotine and ADHD, we examined nicotine's effects on functional brain networks in an animal model of ADHD. Awake magnetic resonance imaging was used to compare functional connectivity in adolescent (post-natal day 44 ± 2) males of the spontaneously hypertensive rat (SHR) strain and two control strains, Wistar-Kyoto and Sprague-Dawley (n = 16 each). We analyzed functional connectivity immediately before and after nicotine exposure (0.4 mg/kg base) in naïve animals, using a region-of-interest approach focussing on 16 regions previously implicated in reward and addiction. Relative to the control groups, the SHR strain demonstrated increased functional connectivity between the ventral tegmental area (VTA) and retrosplenial cortex in response to nicotine, suggesting an aberrant response to nicotine. In contrast, increased VTA-substantia nigra connectivity in response to a saline injection in the SHR was absent following a nicotine injection, suggesting that nicotine normalized function in this circuit. In the SHR, nicotine triggered an atypical response in one VTA circuit while normalizing activity in another. The VTA has been widely implicated in drug reward. Our data suggest that increased susceptibility to nicotine addiction in individuals with ADHD may involve altered responses to nicotine involving VTA circuits. Nicotine addiction is more common among individuals with ADHD. We found that two circuits involving the VTA responded differently to nicotine in animals that model ADHD in comparison to two control strains. In one circuit, nicotine normalized activity that was abnormal in the ADHD animals, while in the other circuit nicotine caused an atypical brain response in the ADHD animals. The VTA has been implicated in drug reward. Our results would be consistent with an interpretation that nicotine may normalize abnormal brain activity in ADHD, and that nicotine may be more rewarding for individuals with ADHD.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We have developed a novel \"2-hit\" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57BL6/NJ mice fed a high fat diet for >10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse . Control mice, HFD only, Renin only and HFD-Renin (aka \"HFpEF\") littermates underwent a battery of cardiac and extracardiac phenotyping. HFD-Renin mice demonstrated obesity and insulin resistance, a 2-3-fold increase in circulating renin levels that resulted in 30-40% increase in left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by altered E/e', IVRT, and strain measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to findings in human HFpEF. Treatment of these mice with the sodium-glucose cotransporter 2 inhibitor empagliflozin, an effective but incompletely understood HFpEF therapy, improved exercise tolerance, left heart enlargement, and insulin homeostasis. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Addition of HFD-Renin mice to the preclinical HFpEF model platform allows for orthogonal studies to increase validity in assessment of interventions. Heart failure with preserved ejection fraction (HFpEF) is a complex disease to study due to limited preclinical models. We rigorously characterize a new two-hit HFpEF mouse model, which allows for dissecting individual contributions and synergy of major pathogenic drivers, hypertension and diet-induced obesity. The results are consistent and reproducible in two independent laboratories. This high-fidelity pre-clinical model increases the available, orthogonal models needed to improve our understanding of the causes and assessment treatments for HFpEF.

Circulating triglycerides are principally transported by triglyceride-rich lipoprotein particles (TRLs) including very-low-density-lipoproteins (VLDL) and chylomicrons and require the activity of lipoprotein lipase for appropriate lipid processing and cellular uptake. Despite known genetic links between CD300LG variants and altered lipid profiles, the functional role of CD300LG in lipid metabolism remains unclear. In this study, we identify CD300LG as a crucial receptor for TRLs. Human genetic analyses reveal that reduced CD300LG protein levels are causally linked with CAD risk and increased number, diameter, and TAG concentration of TRLs. In mice, CD300LG deficiency results in postprandial hypertriglyceridemia independent of changes in VLDL secretion, intestinal lipid absorption, or lipoprotein lipase activity. Mechanistically, CD300LG acts as a receptor for TRLs through a direct interaction with ApoA4 to facilitate TRL clearance at the microvascular endothelium. These findings elucidate new functions for both CD300LG and ApoA4 and advance our general understanding of triglyceride metabolism.

In 1939 he persuaded his two most promising graduate students, Hewlett and Packard, to form a company for making scientific instruments and test equipment. The Hewlett-Packard Company - HP for short - was founded on the principles of constant innovation, openness and employee participation that now define the Silicon Valley business culture. Today, HP is Silicon Valley's largest employer. It has sales approaching $50bn and 88,500 employees world- wide. William Redington Hewlett was born in Ann Arbor, Michigan, in 1913, moving to California in 1916, when his physician father joined the faculty of Stanford Medical School. As a child, Hewlett was fascinated by science and electronic gadgetry, which led him to enrol on the engineering programme at Stanford in 1930. There he struck up a friendship with his classmate [David Packard] that was to last over 60 years until Packard's death in 1996. Hewlett and Packard briefly left California for careers in more promising surroundings, but were lured back by [Frederick Terman] to complete their graduate studies at Stanford. In 1939, besides forming the Hewlett-Packard Company, Hewlett married Flora Lamson, a biochemist, with whom he went on to have five children. During the war years, while Packard ran the company, Hewlett served as an engineer in the US Army Signal Corps. On his return from the war, and for the next 30 years, he served alongside Packard in both technical and managerial positions. Of the partners, Packard has been described as the entrepreneur-manager and Hewlett the entrepreneur-inventor, although each was capable of both managerial and technical roles.

Intrinsically disordered regions (IDRs) are prevalent in RNA-binding proteins (RBPs), yet their roles in RNA interactions remain poorly defined. We examined RNA-binding regulation by structured and disordered regions of LARP6, an RBP with a diverse RNA-binding repertoire. Mass spectrometry-based RNA interaction mapping in living cells identified direct LARP6–RNA contacts within the structured La-module and its flanking IDRs. Mutagenesis and individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) revealed the La-module, but not the IDRs, as essential for LARP6 RNA binding. Deletion of the N-terminal IDR broadened LARP6 RNA footprints, uncovering a role in RNA-binding selectivity. This is achieved through a composite mechanism of restricting the conformational flexibility of the adjacent La-module, forming auxiliary contacts with the RNA, and modulating RNA access for binding. The IDR-mediated RNA-binding selectivity is critical for LARP6-mediated promotion of cancer cell viability and invasion. Our findings uncover a previously unrecognised critical function for IDRs in promoting selective RBP–RNA recognition, by affecting the binding specificity of their adjacent structured domains. LARP6 is an EMT-associated RNA-binding protein with diverse RNA targets. Here, the authors show that the N-terminal disordered region of LARP6 promotes RNA-binding selectivity by modulating the adjacent La-module and forming auxiliary contacts with RNA.