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Insulin resistance dysregulates CYP7B1 leading to oxysterol accumulation: a pathway for NAFL to NASH transition [Research Articles]

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.




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The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis [Research Articles]

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.




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Stimulation of ABCB4/MDR3 ATPase activity requires an intact phosphatidylcholine lipid [Research Articles]

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.




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SCD1 promotes lipid mobilization in subcutaneous white adipose tissue [Research Articles]

Beiging of white adipose tissue (WAT) has beneficial effects on metabolism. Although it is known that beige adipocytes are active in lipid catabolism and thermogenesis, how they are regulated deserves more explorations. In this study, we demonstrate that stearoyl-CoA desaturase 1 (SCD1) in subcutaneous WAT (scWAT) responded to cold stimulation and was able to promote mobilization of triacylglycerol [TAG (triglyceride)]. In vitro studies showed that SCD1 promoted lipolysis in C3H10T1/2 white adipocytes. The lipolytic effect was contributed by one of SCD1’s products, oleic acid (OA). OA upregulated adipose TAG lipase and hormone-sensitive lipase expression. When SCD1 was overexpressed in the scWAT of mice, lipolysis was enhanced, and oxygen consumption and heat generation were increased. These effects were also demonstrated by the SCD1 knockdown experiments in mice. In conclusion, our study suggests that SCD1, known as an enzyme for lipid synthesis, plays a role in upregulating lipid mobilization through its desaturation product, OA.




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Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood [Research Articles]

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1–/– mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.




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Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice [Research Articles]

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp–/–) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp–/– mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp–/– mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp–/– mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp–/– mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp–/– mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp–/– mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp–/– mice.




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{alpha}-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate [Membrane Biology]

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P2 levels on the plasma membrane. In accord with their effects on PI(4,5)P2 levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P2 levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.




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The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint [Research]

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1. Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.




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High-dimensional Cytometry (ExCYT) and Mass Spectrometry of Myeloid Infiltrate in Clinically Localized Clear Cell Renal Cell Carcinoma Identifies Novel Potential Myeloid Targets for Immunotherapy [Research]

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers worldwide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment-naïve clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45+) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.




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Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals [Research]

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.




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Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry [Research]

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.




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Detection of multiple autoantibodies in patients with ankylosing spondylitis using nucleic acid programmable protein arrays [11. Microarrays/Combinatorics/Display Technology]

Ankylosing Spondylitis (AS) is a common, inflammatory rheumatic disease, which primarily affects the axial skeleton and is associated with sacroiliitis, uveitis and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine if plasma from patients with AS contained autoantibodies and if so, characterize and quantify this response in comparison to patients with Rheumatoid Arthritis (RA) and healthy controls. Two high-density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis in order to determine patterns of signalling cascades or tissue origin. 44% of patients with Ankylosing Spondylitis demonstrated a broad autoantibody response, as compared to 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The AS patients autoantibody responses were targeted towards connective, skeletal and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic Acid Programmable Protein Arrays constitute a powerful tool to study autoimmune diseases.




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Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development [Research]

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.




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A proteomic approach to understand the clinical significance of acute myeloid leukemia-derived extracellular vesicles reflecting essential characteristics of leukemia [Research]

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. Enzyme-linked immunosorbent assay results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML-derived EVs could reflect essential leukemia biology.




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Nuclear Disarmament and the Protection of Cultural Heritage

Nuclear Disarmament and the Protection of Cultural Heritage Research paper sysadmin 6 October 2017

States possessing nuclear weapons should be called upon to consider and publish the risks posed to cultural heritage, and their mitigation strategies, in their nuclear-weapons doctrines and policies.

A woman walks on the roof of the Great Mosque of Djenné, a World Heritage Site, after praying. Photo: United Nations.

Summary

  • Renewed risk assessments for nuclear weapons and policies are taking place around the world in light of nuclear modernization and the changing geostrategic environment that is making the use of nuclear weapons more likely. As such the humanitarian impacts of nuclear weapons and tests have received increased attention. However, the effect on cultural heritage has so far been neglected.
  • The potential for armed conflict to destroy cultural heritage has been recognized in international law since 1954. There is significant evidence on the impact of nuclear weapons on cultural heritage including the consequences of their use in Hiroshima and Nagasaki and the effect of nuclear-testing programmes in places of cultural significance since 1945. States that possess nuclear weapons have increased liabilities and responsibilities to protect cultural heritage and cultural rights. The need to protect cultural heritage should strengthen the case for reducing and eliminating nuclear weapons.
  • Failure to take into account the protection of heritage in the development of nuclear weapons policies – including disarmament, non-proliferation and arms-control negotiations – significantly undermines states’ existing commitments to protecting heritage threatened by conflict.
  • Risk assessments of the impact of nuclear weapons on cultural heritage and important cultural artefacts – and methods of preventing such catastrophic damage – should be part of protecting cultural heritage in every country and the subject of informed public debate. A new body of knowledge on the full range of nuclear weapons impacts would introduce a fresh perspective to inform decision-makers, international organizations and the public in thinking about nuclear weapons policies and practices.
  • Risk and resilience frameworks, which provide sets of solutions for risk assessments, would allow assessments of nuclear weapons threats to heritage and highlight vulnerabilities that need to be addressed. Such frameworks would provide a basis for policymakers to identify the world’s cultural heritage most at risk and help develop mitigation strategies to ensure that it is protected. In particular, states possessing nuclear weapons should be called upon to consider and publish the risks posed to cultural heritage, and their mitigation strategies, in their nuclear weapons doctrines and policies, as a contribution to transparency and confidence-building, and as a responsibility to the world’s shared heritage. International organizations, such as the UN Educational, Scientific, and Cultural Organization (UNESCO), have a role to play in bridging security perspectives with protecting cultural heritage.




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Mycobacteria excise DNA damage in 12- or 13-nucleotide-long oligomers by prokaryotic-type dual incisions and performs transcription-coupled repair [Genomics and Proteomics]

In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual-incision patterns have been discovered: the prokaryotic type, which removes the damage in 11–13-nucleotide-long oligomers, and the eukaryotic type, which removes the damage in 24–32-nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nt from the 3´ end and 9 nt from the 5´ end flanking the damage. To test this model, we used the in vivo excision assay and the excision repair sequencing genome-wide repair mapping method developed in our laboratory to determine the repair pattern and genome-wide repair map of Mycobacterium smegmatis. We find that M. smegmatis, which possesses homologs of the Escherichia coli uvrA, uvrB, and uvrC genes, removes cyclobutane pyrimidine dimers from the genome in a manner identical to the prokaryotic pattern by incising 7 nt 5´ and 3 or 4 nt 3´ to the photoproduct, and performs transcription-coupled repair in a manner similar to E. coli.




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Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ϵ holoenzyme [DNA and Chromosomes]

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.




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Mine Action in Angola: Clearing the Legacies of Conflict to Harness the Potential of Peace

Mine Action in Angola: Clearing the Legacies of Conflict to Harness the Potential of Peace Other resource sysadmin 14 June 2019

This publication draws on and updates the briefing note published following a meeting of the All- Party Parliamentary Group (APPG) on Angola on 26 April 2017. It also incorporates insights from a Chatham House Africa Programme conference session on the legacies of the Angolan Civil War, held on 23 March 2018; and draws on the Africa Programme’s research into conservation-driven development models in Southern Africa.

A mine clearance specialist in Angola preparing equipment used to look for unexploded ordnance, May 2012. Photo: Eye Ubiquitous/Contributor/Getty Images.

Almost two decades after the end of its civil war, Angola remains one of the most heavily landmine-contaminated countries in the world. The Angolan government has committed to clearing its landmines by 2025, and there is constructive collaboration between the government and mine clearing agencies in this endeavour, but the target will be achievable only if a decline in funding from international donors is reversed. International funding for mine clearance in Angola fell by more than 80 per cent between 2005 and 2017, and this sharp drop in external support has compounded the impact on domestic funding for national clearance efforts as a result of the downturn in prices for Angola’s main export commodities.

The national mine action agency, the Comissão Nacional Intersectorial de Desminagem e Assistência Humanitária (CNIDAH), is supported by the Mines Advisory Group (MAG), Norwegian People’s Aid (NPA) and the HALO Trust. By 2017, 15 years after the end of the civil war, these organizations had collectively helped clear 56 per cent of known landmine-contaminated land. State-led demining has focused principally on clearing areas designated for infrastructure projects. Now, it is critical that humanitarian demining in largely agricultural and conservation areas is prioritized to bring to an end the daily threat to Angola’s rural poor – as well as to the country’s livestock and wildlife – of injury or death as a result of landmine accidents.

Angola has some of the world’s most important remaining wilderness, including the tributary system for the unique Okavango Delta, and the country has the potential to host one of the most diverse wildlife populations on the continent. However, the presence of landmines and other remnants of the civil war render large areas of the country unsafe both for wildlife and for the local people, whose ability to derive a sustainable livelihood from their natural environment is fundamental to its protection.

Wildlife and tourism provide important economic opportunities for diversification beyond an oil-dominated economy. Critically, Angola’s economic diversification and development objectives can only be achieved if the landmines that prohibit access to land for agriculture, mining, tourism and wildlife are cleared.

There are economic opportunities for released land in the most heavily mined provinces of Cuando Cubango and Moxico. Already, some new funding for mine action in Angola, if upscaled or matched by international donors, could be transformative for its people, and for the conservation of the region’s vital biodiversity.




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Endovascular treatment for acute ischaemic stroke in routine clinical practice: prospective, observational cohort study (MR CLEAN Registry)




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A new nuclear order

A new nuclear order 7 February 2023 — 6:00PM TO 7:00PM Anonymous (not verified) 26 January 2023 Chatham House and Online

In conversation with Rafael Mariano Grossi.

For more than half a century, the global nuclear non-proliferation framework has supported international security and facilitated the expansion of the many peaceful applications of nuclear science and technology. 

What is happening today in Ukraine, Iran and North Korea, not only challenges the way we deal with the existential threat of nuclear weapons, but also the impact it could have on addressing another existential threat – climate change.

Russia’s invasion of Ukraine is the biggest test to global resolve both in avoiding nuclear conflict and in ensuring the safety of one of the biggest nuclear power programmes in Europe.

Rafael Mariano Grossi of the International Atomic Energy Agency discusses key questions on global nuclear cooperation including:

  • The impact of the war in Ukraine and issues with Iran and North Korea on countries’ risk assessment with regards to nuclear non-proliferation.

  • What the IAEA’s on-the-ground presence and the director general’s missions to Ukraine, particularly the Zaporizhzhia Nuclear Power Plant, tells us about what is necessary – now and in the long term – to ensure the safety and security of nuclear material under all circumstances.

  • The role of ensuring nuclear energy can play its vital part in mitigating climate change now and in the future.

As with all member events, questions from the audience drive the conversation.

Read the transcript.




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[68Ga]Ga-PSMA-11 PET/CT-Positive Hepatic Inflammatory Pseudotumor: Possible PSMA-Avid Pitfall in Nuclear Imaging




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The Emission of Internal Conversion Electrons Rather Than Auger Electrons Increased the Nucleus-Absorbed Dose for 161Tb Compared with 177Lu with a Higher Dose Response for [161Tb]Tb-DOTA-LM3 Than for [161Tb]Tb-DOTATATE

Preclinical data have shown that 161Tb-labeled peptides targeting the somatostatin receptor are therapeutically more effective for peptide receptor radionuclide therapy than are their 177Lu-labeled counterparts. To further substantiate this enhanced therapeutic effect, we performed cellular dosimetry to quantify the absorbed dose to the cell nucleus and compared dose–response curves to evaluate differences in relative biological effectiveness in vitro. Methods: CA20948 cell survival was assessed after treatment with [161Tb]Tb- and [177Lu]Lu-DOTATATE (agonist) and with [161Tb]Tb- and [177Lu]Lu-DOTA-LM3 (antagonist) via a clonogenic assay. Cell binding, internalization, and dissociation assays were performed up to 7 d to acquire time-integrated activity coefficients. Separate S values for each type of particle emission (Auger/internal conversion [IC] electrons and β particles) were computed via Monte Carlo simulations, while considering spheric cells. Once the absorbed dose to the cell nucleus was calculated, survival curves were fitted to the appropriate linear or linear-quadratic model and corresponding relative biological effectiveness was evaluated. Results: Although the radiopeptide uptake was independent of the radionuclide, [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 delivered a 3.6 and 3.8 times higher dose to the nucleus, respectively, than their 177Lu-labeled counterparts on saturated receptor binding. This increased nucleus-absorbed dose was mainly due to the additional emission of IC and not Auger electrons by 161Tb. When activity concentrations were considered, both [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 showed a lower survival fraction than did labeling with 177Lu. When the absorbed dose to the nucleus was considered, no significant difference could be observed between the dose–response curves for [161Tb]Tb- and [177Lu]Lu-DOTATATE. [161Tb]Tb-DOTA-LM3 showed a linear-quadratic dose response, whereas [161Tb]Tb-DOTATATE showed only a linear dose response within the observed dose range, suggesting additional cell membrane damage by Auger electrons. Conclusion: The IC, rather than Auger, electrons emitted by 161Tb resulted in a higher absorbed dose to the cell nucleus and lower clonogenic survival for [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 than for the 177Lu-labeled analogs. In contrast, [161Tb]Tb-DOTATATE showed no higher dose response than [177Lu]Lu-DOTATATE, whereas for [161Tb]Tb-DOTA-LM3 an additional quadratic response was observed. Because of this quadratic response, potentially caused by cell membrane damage, [161Tb]Tb-DOTA-LM3 is a more effective radiopeptide than [161Tb]Tb-DOTATATE for labeling with 161Tb.




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Nuclear Imaging of Bispecific Antibodies on the Rise

Bispecific antibodies (bsAbs) are engineered to target 2 different epitopes simultaneously. About 75% of the 16 clinically approved bsAbs have entered the clinic internationally since 2022. Hence, research on biomedical imaging of various radiolabeled bsAb scaffolds may serve to improve patient selection for bsAb therapy. Here, we provide a comprehensive overview of recent advances in radiolabeled bsAbs for imaging via PET or SPECT. We compare direct targeting and pretargeting approaches in preclinical and clinical studies in oncologic research. Furthermore, we show preclinical applications of imaging bsAbs in neurodegenerative diseases. Finally, we offer perspectives on the future directions of imaging bsAbs based on their challenges and opportunities.




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U.S., South Korea agree to greater cooperation on civil nuclear energy

Earlier this month, the United States and the Republic of Korea reached an agreement on greater cooperation on civil nuclear energy projects, the U.S. Department of Energy said this week.




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Google Cloud and Oracle Veteran Amit Zavery to Spearhead Product and Engineering at ServiceNow

SANTA CLARA, Calif., Oct. 25, 2024 — ServiceNow has announced that enterprise software industry veteran Amit Zavery will join the company as president, chief product officer (CPO), and chief operating officer […]

The post Google Cloud and Oracle Veteran Amit Zavery to Spearhead Product and Engineering at ServiceNow appeared first on HPCwire.




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Watch: British daredevil dragged behind motorcycle at 159 mph

A British daredevil was intentionally dragged behind his motorcycle at a speed of 159.52 mph to break a Guinness World Record.





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Wendi McLendon-Covey, David Alan Grier, Allison Tolman enjoying flawed 'St. Denis' roles

Wendi McLendon-Covey, David Alan Grier and Allison Tolman discuss the comedy in their harried medical characters on "St. Denis Medical," premiering Tuesday on NBC.




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35 dead, 43 injured in vehicle attack at sports center in China

A man drove a vehicle into a crowd at a sports center in Zhuhai, China, killing 35 people and injuring 43 others, police said.




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Google educates users about the moon cycle with new Doodle game

Google is educating users about the lunar cycle with its latest interactive Doodle released Thursday.




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This Technique Allows Chronic Worriers To Think More Clearly

A simple 8-minute exercise can free up your brain and make you more efficient under stress.



  • Boost Brain Power

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Ohio House OKs school funding plan; unclear if Senate will




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Ohio House OKs school funding plan; unclear if Senate will




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Clemson makes a small move up the latest College Football Playoff rankings

The College Football Playoff Selection Committee has released its second set of rankings, and Clemson football's moved up to No. 20 following a 24-14 win over Virginia Tech. Starting at No. 23 in last week’s CFP rankings, the Tigers’ latest rise highlights thei




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Extracellular Vesicle-Mediated Neuron-Glia Communications in the Central Nervous System

Tsuneya Ikezu
Oct 2, 2024; 44:e1170242024-e1170242024
Symposium




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Neuregulin1 Nuclear Signaling Influences Adult Neurogenesis and Regulates a Schizophrenia Susceptibility Gene Network within the Mouse Dentate Gyrus

Prithviraj Rajebhosale
Oct 23, 2024; 44:e0063242024-e0063242024
Cellular




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Brief and Diverse Excitotoxic Insults Increase the Neuronal Nuclear Membrane Permeability in the Neonatal Brain, Resulting in Neuronal Dysfunction and Cell Death

Neuronal cytotoxic edema is implicated in neuronal injury and death, yet mitigating brain edema with osmotic and surgical interventions yields poor clinical outcomes. Importantly, neuronal swelling and its downstream consequences during early brain development remain poorly investigated, and new treatment approaches are needed. We explored Ca2+-dependent downstream effects after neuronal cytotoxic edema caused by diverse injuries in mice of both sexes using multiphoton Ca2+ imaging in vivo [Postnatal Day (P)12–17] and in acute brain slices (P8–12). After different excitotoxic insults, cytosolic GCaMP6s translocated into the nucleus after a few minutes in a subpopulation of neurons, persisting for hours. We used an automated morphology-detection algorithm to detect neuronal soma and quantified the nuclear translocation of GCaMP6s as the nuclear to cytosolic intensity (N/C ratio). Elevated neuronal N/C ratios occurred concurrently with persistent elevation in Ca2+ loads and could also occur independently from neuronal swelling. Electron microscopy revealed that the nuclear translocation was associated with the increased nuclear pore size. The nuclear accumulation of GCaMP6s in neurons led to neocortical circuit dysfunction, mitochondrial pathology, and increased cell death. Inhibiting calpains, a family of Ca2+-activated proteases, prevented elevated N/C ratios and neuronal swelling. In summary, in the developing brain, we identified a calpain-dependent alteration of nuclear transport in a subpopulation of neurons after disease-relevant insults leading to long-term circuit dysfunction and cell death. The nuclear translocation of GCaMP6 and other cytosolic proteins after acute excitotoxicity can be an early biomarker of brain injury in the developing brain.




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Neuregulin1 Nuclear Signaling Influences Adult Neurogenesis and Regulates a Schizophrenia Susceptibility Gene Network within the Mouse Dentate Gyrus

Neuregulin1 (Nrg1) signaling is critical for neuronal development and function from fate specification to synaptic plasticity. Type III Nrg1 is a synaptic protein which engages in bidirectional signaling with its receptor ErbB4. Forward signaling engages ErbB4 phosphorylation, whereas back signaling engages two known mechanisms: (1) local axonal PI3K-AKT signaling and (2) cleavage by -secretase resulting in cytosolic release of the intracellular domain (ICD), which can traffic to the nucleus (Bao et al., 2003; Hancock et al., 2008). To dissect the contribution of these alternate signaling strategies to neuronal development, we generated a transgenic mouse with a missense mutation (V321L) in the Nrg1 transmembrane domain that disrupts nuclear back signaling with minimal effects on forward signaling or local back signaling and was previously found to be associated with psychosis (Walss-Bass et al., 2006). We combined RNA sequencing, retroviral fate mapping of neural stem cells, behavioral analyses, and various network analyses of transcriptomic data to investigate the effect of disrupting Nrg1 nuclear back signaling in the dentate gyrus (DG) of male and female mice. The V321L mutation impairs nuclear translocation of the Nrg1 ICD and alters gene expression in the DG. V321L mice show reduced stem cell proliferation, altered cell cycle dynamics, fate specification defects, and dendritic dysmorphogenesis. Orthologs of known schizophrenia (SCZ)-susceptibility genes were dysregulated in the V321L DG. These genes coordinated a larger network with other dysregulated genes. Weighted gene correlation network analysis and protein interaction network analyses revealed striking similarity between DG transcriptomes of V321L mouse and humans with SCZ.




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Retinal Input to Macaque Superior Colliculus Derives from Branching Axons Projecting to the Lateral Geniculate Nucleus

The superior colliculus receives a direct projection from retinal ganglion cells. In primates, it remains unknown if the same ganglion cells also supply the lateral geniculate nucleus. To address this issue, a double-label experiment was performed in two male macaques. The animals fixated a target while injection sites were scouted in the superior colliculus by recording and stimulating with a tetrode. Once suitable sites were identified, cholera toxin subunit B-Alexa Fluor 488 was injected via an adjacent micropipette. In a subsequent acute experiment, cholera toxin subunit B-Alexa Fluor 555 was injected into the lateral geniculate nucleus at matching retinotopic locations. After a brief survival period, ganglion cells were examined in retinal flatmounts. The percentage of double-labeled cells varied locally, depending on the relative efficiency of retrograde transport by each tracer and the precision of retinotopic overlap of injection sites in each target nucleus. In counting boxes with extensive overlap, 76–98% of ganglion cells projecting to the superior colliculus were double labeled. Cells projecting to the superior colliculus constituted 4.0–6.7% of the labeled ganglion cell population. In one particularly large zone, there were 5,746 cells labeled only by CTB-AF555, 561cells double labeled by CTB-AF555 and CTB-AF488, but no cell labeled only by CTB-AF488. These data indicate that retinal input to the macaque superior colliculus arises from a collateral axonal branch supplied by ~5% of the ganglion cells that project to the lateral geniculate nucleus. Surprisingly, there exist no ganglion cells that project exclusively to the SC.




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Erratum: McCosh et al., "Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice"




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MIT's New Water-Powered Artificial Muscles

Scientists have created thin polymer sheets that expand and contract when in contact with water




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A Multimedia Spectacle at the Hirshhorn

Read more about SONG 1: http://j.mp/GTZ0Ru & http://j.mp/GVlcMx Watch how artist Doug Aitken transforms the outside of the art museum into a panoramic movie screen




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The Stunningly Clear Features on the Lindow Man Mummy

Lindow Man, believed to be a victim of human sacrifice, remains one of the best preserved ancient bodies in all of Europe. The level of detail on his face is staggering—not bad for a 2,000-year-old mummy




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A Cycle Opera Rehearsal

Theater students in Scunthorpe, England, rehearse for their performance celebrating the life of homegrown cyclist Lal White




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What Roberto Clemente Meant to Baseball

Biographer David Maraniss says that in order to truly understand Clemente's importance to the sport, you have to look beyond his spectacular numbers




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World's First Ultra-Precise Nuclear Clock Is Within Reach After Major Breakthrough, Researchers Say

The technology, enabled by thorium atoms, could keep time more accurately than atomic clocks and enable new discoveries about gravity, gravitational waves and dark matter




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Rome's Trevi Fountain Will Get a Much-Needed Cleaning—and a Controversial New Entry Fee

During the restorations, visitors will be able to see the famous site via a temporary walkway, which officials will use to study the flow of foot traffic




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Nobel Peace Prize Goes to Japanese Atomic Bomb Survivors Who Fight for Nuclear Disarmament

The grassroots organization, Nihon Hidankyo, was lauded for "demonstrating through witness testimony that nuclear weapons must never be used again"




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Cape Breton woman tracks down great-uncle's lost WWII medals in time for Remembrance Day

A Port Hawkesbury woman has long imagined a Remembrance Day with her great-uncle's war medals. It will happen this year for the first time thanks to some sleuthing and the kindness of her great-uncle's foster family.



  • News/Canada/Nova Scotia

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RCMP investigating after body found in burned vehicle in Pictou County

RCMP are investigating after officers discovered human remains in an abandoned, burned vehicle in Greenhill, N.S.



  • News/Canada/Nova Scotia