Taiwanese network-attached storage (NAS) appliance maker Synology has addressed a critical security flaw impacting DiskStation and BeePhotos that could lead to remote code execution. Tracked as CVE-2024-10443 and dubbed RISK:STATION by Midnight Blue, the zero-day flaw was demonstrated at the Pwn2Own Ireland 2024 hacking contest by security researcher Rick de Jager. RISK:STATION is an "

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment–protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

In their comment (1) on our publication (2), the authors make two points: (i) they raise concerns about the possible effect of residual NONOate in our study, and (ii) they promote nitrosylcobalamin (NOCbl) supplied by their own company. Both points lack merit for the following reasons. The authors make the astonishing claim that the spectra of nitric oxide (NO•) and cobalamins overlap. Unlike NO•, cobalamin absorbs in the visible region, permitting unequivocal spectral assignment of NOCbl as reported (3). We demonstrated that whereas NOCbl is highly unstable in solution, it is stabilized by the B12 trafficking protein CblC. So even if present, residual NONOate (which is unstable at neutral pH and is removed during the work-up (3)) could not account for the observed difference.The authors then misrepresent our synthetic method, claiming that anaerobic conditions were used to generate nitrocobalamin (NO2Cbl), which results in the transient formation of NOCbl. We synthesized NO2Cbl aerobically using nitrite as described (4); NOCbl is not an intermediate in this ligand exchange reaction. The aerobic instability of NOCbl has been rigorously described by inorganic chemists (3, 5) and raises obvious questions about its purported biological effects as exemplified by the authors' own 2003 JBC publication, which was later withdrawn.As to promoting NOCbl from their company, the authors refer to a synthetic route from a mixture of NO• gas and aquocobalamin. The authors' method (6) has been described as “dubious” by chemists (5). Whereas DEAE NONOate used in our method is widely known as an NO• donor,...

After a thorough read of this paper (1), we wish to clarify that the authors' anaerobic method of synthesis for the production of nitrocobalamin results in the transient formation of nitrosylcobalamin, an unstable intermediate upon exposure to air. We concur that the authors' method results in the production of nitrocobalamin based on the UV-visible data as shown. The authors' adapted anaerobic method consists of mixing hydroxocobalamin hydrochloride with diethylamine NONOate diethylammonium salt in aqueous solution. Of concern, the UV spectrum of nitric oxide overlaps that of all cobalamin species under anaerobic conditions, making any assignments of the binding of nitric oxide to hydroxocobalamin suspect (2). Additionally, the use of acetone to precipitate the authors' product causes precipitation of diethylamine NONOate, resulting in an impure product. As a result, its utility for drawing experimental conclusions is faulty.The product from the authors' anaerobic synthetic method has not been assessed for purity, and the synthetic method itself has not been validated using a stability-indicating method as required by the International Conference on Harmonization (ICH) (ICH Q2B, Validation of Analytical Procedures) methodology, which is a hallmark for analytical characterization. Our nitrosylcobalamin synthesis involves reacting nitric oxide gas with hydroxocobalamin acetate as a heterogeneous mixture in a non-electron-donating solvent followed by rotary evaporation. Our nitrosylcobalamin product is stable in air, releases nitric oxide gas in situ (3), and meets ICH stability guidelines (4). Additionally, our nitrosylcobalamin product demonstrates biological activity, which has not been observed for nitrocobalamin (3, 5).

Dongmei Cheng
Jun 1, 2006; 5:1158-1170
Datasets

Jonathan C. Trinidad
Aug 1, 2012; 11:215-229
Research

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether–containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during Cα-thioether bridge LC–MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Margrit Schwarz
Sep 1, 1998; 39:1833-1843
Articles

MS Brown
Jul 1, 1980; 21:505-517
Reviews

Chi-Liang Eric Yen
Nov 1, 2008; 49:2283-2301
Thematic Reviews

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15–25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host–pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week–old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75–Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Hideaki Kuge
Dec 1, 2020; 61:1747-1763
Research Articles

Babunageswararao Kanuri
Nov 17, 2020; 0:jlr.RA120001101v1-jlr.RA120001101
Research Articles

This manuscript has been withdrawn by the Author.

Mendelian randomization (MR) of lipid traits in coronary artery disease (CAD) has provided evidence for causal associations of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in CAD, but many lipid trait genetic variants have pleiotropic effects on other cardiovascular risk factors that may bias MR associations. The goal of this study was to evaluate pleiotropic effects of lipid trait genetic variants and to account for these effects in MR of lipid traits in CAD. We performed multivariable MR using inverse variance-weighted (IVW) and MR-Egger methods in large (n ≥ 300,000) GWAS datasets. We found that 30% of lipid trait genetic variants have effects on metabolic syndrome traits, including body mass index (BMI), type 2 diabetes (T2D), and systolic blood pressure (SBP). Nonetheless, in multivariable MR analysis, LDL-C, high-density lipoprotein cholesterol (HDL-C), TG, BMI, T2D, and SBP are independently associated with CAD, and each of these associations is robust to adjustment for directional pleiotropy. MR at loci linked to direct effects on HDL-C and TG suggests locus- and mechanism-specific causal effects of these factors on CAD.

Smith-Lemli-Opitz Syndrome (SLOS) is a developmental disorder (OMIM #270400) caused by autosomal recessive mutations in the Dhcr7 gene, which encodes the enzyme 3β-hydroxysterol-7 reductase. SLOS patients present clinically with dysmorphology and neurological, behavioral and cognitive defects, with characteristically elevated levels of 7-dehydrocholesterol (7-DHC) in all bodily tissues and fluids. Previous mouse models of SLOS have been hampered by postnatal lethality when Dhcr7 is knocked out globally, while a hypomorphic mouse model showed improvement in the biochemical phenotype with ageing, and did not manifest most other characteristic features of SLOS. We report the generation of a conditional knockout of Dhcr7 (Dhcr7flx/flx), validated by generating a mouse with a liver-specific deletion (Dhcr7L-KO). Phenotypic characterization of liver-specific knockout mice revealed no significant changes in viability, fertility, growth curves, liver architecture, hepatic triglyceride secretion, or parameters of systemic glucose homeostasis. Furthermore, qPCR and RNA-Seq analyses of livers revealed no perturbations in pathways responsible for cholesterol synthesis, either in male or female Dhcr7L-KO mice, suggesting hepatic disruption of post-squalene cholesterol synthesis leads to minimal impact on sterol metabolism in the liver. This validated conditional Dhcr7 knockout model may now allow us to systematically explore the pathophysiology of SLOS, by allowing for temporal, cell and tissue-specific loss of DHCR7.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter allopregnanolone (AlloP) levels, an important neurosteroid in the brain. We assessed the levels and regulation of AlloP and the effects of AlloP supplementation on cognitive function in 4-month-old and 24-month-old male C57BL/6 mice. With age, the expression of enzymes involved in the AlloP synthetic pathway was decreased and corticosterone (CORT) synthesis increased. Supplementation of AlloP improved cognitive function. Interestingly, interleukin 6 (IL-6) infusion in young animals significantly reduced the production of AlloP compared with controls. It is notable that inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein 130, significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of AlloP and increases CORT levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in AlloP levels and an increase in CORT. Furthermore, our results indicate that AlloP is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1–CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage–activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.

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.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

The peptide hormone ghrelin is produced in cardiomyocytes and acts through the myocardial growth hormone secretagogue receptor (GHSR) to promote cardiomyocyte survival. Administration of ghrelin may have therapeutic effects on post–myocardial infarction (MI) outcomes. Therefore, there is a need to develop molecular imaging probes that can track the dynamics of GHSR in health and disease to better predict the effectiveness of ghrelin-based therapeutics. We designed a high-affinity GHSR ligand labeled with ¹⁸F for imaging by PET and characterized its in vivo properties in a canine model of MI. Methods: We rationally designed and radiolabeled with ¹⁸F a quinazolinone derivative ([¹⁸F]LCE470) with subnanomolar binding affinity to GHSR. We determined the sensitivity and in vivo and ex vivo specificity of [¹⁸F]LCE470 in a canine model of surgically induced MI using PET/MRI, which allowed for anatomic localization of tracer uptake and simultaneous determination of global cardiac function. Uptake of [¹⁸F]LCE470 was determined by time–activity curve and SUV analysis in 3 regions of the left ventricle—area of infarct, territory served by the left circumflex coronary artery, and remote myocardium—over a period of 1.5 y. Changes in cardiac perfusion were tracked by [¹³N]NH₃ PET. Results: The receptor binding affinity of LCE470 was measured at 0.33 nM, the highest known receptor binding affinity for a radiolabeled GHSR ligand. In vivo blocking studies in healthy hounds and ex vivo blocking studies in myocardial tissue showed the specificity of [¹⁸F]LCE470, and sensitivity was demonstrated by a positive correlation between tracer uptake and GHSR abundance. Post-MI changes in [¹⁸F]LCE470 uptake occurred independently of perfusion tracer distributions and changes in global cardiac function. We found that the regional distribution of [¹⁸F]LCE470 within the left ventricle diverged significantly within 1 d after MI and remained that way throughout the 1.5-y duration of the study. Conclusion: [¹⁸F]LCE470 is a high-affinity PET tracer that can detect changes in the regional distribution of myocardial GHSR after MI. In vivo PET molecular imaging of the global dynamics of GHSR may lead to improved GHSR-based therapeutics in the treatment of post-MI remodeling.

Guo-qiang Bi
Dec 15, 1998; 18:10464-10472
Articles

Robbert Havekes
Dec 12, 2012; 32:18137-18149
BehavioralSystemsCognitive

α-Neurexins are essential and highly expressed presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders. Despite their importance, how the elaborate extracellular sequences of α-neurexins contribute to synapse function is poorly understood. We recently characterized the presynaptic gain-of-function phenotype caused by a missense mutation in an evolutionarily conserved extracellular sequence of neurexin-3α (A687T) that we identified in a patient diagnosed with profound intellectual disability and epilepsy. The striking A687T gain-of-function mutation on neurexin-3α prompted us to systematically test using mutants whether the presynaptic gain-of-function phenotype is a consequence of the addition of side-chain bulk (i.e., A687V) or polar/hydrophilic properties (i.e., A687S). We used multidisciplinary approaches in mixed-sex primary hippocampal cultures to assess the impact of the neurexin-3α^A687 residue on synapse morphology, function and ligand binding. Unexpectedly, neither A687V nor A687S recapitulated the neurexin-3α A687T phenotype. Instead, distinct from A687T, molecular replacement with A687S significantly enhanced postsynaptic properties exclusively at excitatory synapses and selectively increased binding to neuroligin-1 and neuroligin-3 without changing binding to neuroligin-2 or LRRTM2. Importantly, we provide the first experimental evidence supporting the notion that the position A687 of neurexin-3α and the N-terminal sequences of neuroligins may contribute to the stability of α-neurexin–neuroligin-1 trans-synaptic interactions and that these interactions may specifically regulate the postsynaptic strength of excitatory synapses.

Injuries to the central nervous system (CNS) can cause severe neurological deficits. Axonal regrowth is a fundamental process for the reconstruction of compensatory neuronal networks after injury; however, it is extremely limited in the adult mammalian CNS. In this study, we conducted a loss-of-function genetic screen in cortical neurons, combined with a Web resource-based phenotypic screen, and identified synaptotagmin 4 (Syt4) as a novel regulator of axon elongation. Silencing Syt4 in primary cultured cortical neurons inhibits neurite elongation, with changes in gene expression involved in signaling pathways related to neuronal development. In a spinal cord injury model, inhibition of Syt4 expression in cortical neurons prevented axonal sprouting of the corticospinal tract, as well as neurological recovery after injury. These results provide a novel therapeutic approach to CNS injury by modulating Syt4 function.

Impaired inhibitory synapse development is suggested to drive neuronal hyperactivity in autism spectrum disorders (ASD) and epilepsy. We propose a novel mechanism by which astrocytes control the development of parvalbumin (PV)-specific inhibitory synapses in the hippocampus, implicating ephrin-B/EphB signaling. Here, we utilize genetic approaches to assess functional and structural connectivity between PV and pyramidal cells (PCs) through whole-cell patch–clamp electrophysiology, optogenetics, immunohistochemical analysis, and behaviors in male and female mice. While inhibitory synapse development is adversely affected by PV-specific expression of EphB2, a strong candidate ASD risk gene, astrocytic ephrin-B1 facilitates PV->PC connectivity through a mechanism involving EphB signaling in PV boutons. In contrast, the loss of astrocytic ephrin-B1 reduces PV->PC connectivity and inhibition, resulting in increased seizure susceptibility and an ASD-like phenotype. Our findings underscore the crucial role of astrocytes in regulating inhibitory circuit development and discover a new role of EphB2 receptors in PV-specific inhibitory synapse development.

Experience- and activity-dependent transcription is a candidate mechanism to mediate development and refinement of specific cortical circuits. Here, we demonstrate that the activity-dependent transcription factor myocyte enhancer factor 2C (MEF2C) is required in both presynaptic layer (L) 4 and postsynaptic L2/3 mouse (male and female) somatosensory (S1) cortical neurons for development of this specific synaptic connection. While postsynaptic deletion of Mef2c weakens L4 synaptic inputs, it has no effect on inputs from local L2/3, contralateral S1, or the ipsilateral frontal/motor cortex. Similarly, homozygous or heterozygous deletion of Mef2c in presynaptic L4 neurons weakens L4 to L2/3 excitatory synaptic inputs by decreasing presynaptic release probability. Postsynaptic MEF2C is specifically required during an early postnatal, experience-dependent, period for L4 to L2/3 synapse function, and expression of transcriptionally active MEF2C (MEF2C-VP16) rescues weak L4 to L2/3 synaptic strength in sensory-deprived mice. Together, these results suggest that experience- and/or activity-dependent transcriptional activation of MEF2C promotes development of L4 to L2/3 synapses. Additionally, MEF2C regulates the expression of many pre- and postsynaptic genes in postnatal cortical neurons. Interestingly, MEF2C was necessary for activity-dependent expression of many presynaptic genes, including those that function in transsynaptic adhesion and neurotransmitter release. This work provides mechanistic insight into the experience-dependent development of specific cortical circuits.

Cardiac MRI of an animal that has undergone photosynthetic therapy. CREDIT: Stanford University School of Medicine, Department of Cardiothoracic Surgery

Cutting days and weeks from design cycle gives Syncroness more time to focus on designing lightweight, high performance products

New findings reveal that mitochondria in nutrient-deprived cells adopt specialised roles to prioritise either energy generation or amino acid synthesis. Led by Dr. Craig Thompson of Memorial Sloan Kettering Cancer Center, the study identified specific mitochondrial subpopulations, allowing cells to maintain critical functions even under stress. These discoveries have implications for understanding how cancer cells survive low-nutrient conditions and how cells repair after injury.

Scientists at Lawrence Berkeley National Laboratory in California are exploring new techniques for creating superheavy elements, focusing on the potential synthesis of “element 120,” also called unbinilium. If successful, this addition could lead to an eighth row on the periodic table. Using ion bombardment, researchers demonstrated a process that may achieve unbinilium by targeting californium with supercharged titanium ions. The approach offers promise, although creating just two atoms could take weeks. This project marks an important step forward in understanding atomic structures and superheavy elements.

Read the in depth Review of Synology DiskStation DS920+ Storage. Know detailed info about Synology DiskStation DS920+ configuration, design and performance quality along with pros & cons, Digit rating, verdict based on user opinions/feedback.

I've never been much of a fan of forecasting approaches to intermittent demand. In situations like intermittent demand (or other areas where we have little hope of reasonably accurate forecasts), my thinking is "why bother?" If we can't expect to solve the problem with forecasting, we need a different approach. [...]

The post Intermittent Demand Forecasting (new book by Boylan and Syntetos) appeared first on The Business Forecasting Deal.

Digital channels open the door to synthetic identity fraud. Luckily, artificial (AI, that is) can defeat synthetic.

The post Synthetic identity fraud: What you can’t see can hurt you appeared first on The Data Roundtable.

Hi everyone,

I'm new to using Innovus and I'm encountering an issue while trying to perform the "init_design" command. My goal is to perform the place and route. Here are the commands I'm using:

``
set init_verilog ./test.v
set init_top_cell TEST
set init_pwr_net {VDD VDD_2 VDD_3}
set init_gnd_net {VSS VSSA}
set init_lef_file { /home/laumecha/uw_openroad_free45/pdk/Drexel-ECEC575/Encounter/NangateOpenCellLibrary/Back_End/lef/NangateOpenCellLibrary.lef}
set init_mmmc_file {./viewDefinition.tcl}
init_design
```

However, I receive the following error:

```
#% Begin Load netlist data ... (date=06/04 12:07:50, mem=1478.7M)
*** Begin netlist parsing (mem=1439.0M) ***
Created 0 new cells from 0 timing libraries.
Reading netlist ...
Backslashed names will retain backslash and a trailing blank character.
**ERROR: (IMPVL-209):   In Verilog file './test.v', check line 16 near the text # for the issue: 'syntax error'.  Update the text accordingly.
Type 'man IMPVL-209' for more detail.
Verilog file './test.v' has errors!  See above.

*** Memory Usage v#1 (Current mem = 1439.027M, initial mem = 634.098M) ***
#% End Load netlist data ... (date=06/04 12:07:50, total cpu=0:00:00.0, real=0:00:00.0, peak res=1478.7M, current mem=1478.7M)
**ERROR: (IMPVL-902):   Failed to read netlist ./test.v. See previous error messages for details.  Resolve the issues and reload the design.
```

However, the file works perfectly in Genus.

It seems there is a syntax error in my Verilog file at line 16, but I'm not sure how to resolve it. Any guidance or suggestions would be greatly appreciated.

Thanks in advance!

I am trying to remove the cdn_loop_breaker cells from the netlist. 
When I tried the below 2 things, genus synthesis tool removing the cdn_loop_breaker cells but while connecting the cdn_loop_breaker cell input to its proper connection, its somehow misleading the connections

Things i tried:
1.  remove_cdn_loop_breaker -instances *cdn_loop_breaker*
then i just ran remove_cdn_loop_breaker  comand without the -instances switch
2. remove_cdn_loop_breaker  
     
both of the above things are not providing the proper connections after removing the loop_breaker_cells

can anyone suggest the best possible workaround for this please?

In a network containing multiple nodes, the need for synchronization between the various nodes is not just instrumental but also a complicated and highly complex process. This process becomes even more tricky if we synchronize the clocks between the Manager and the Peripheral. As we know, in a real-time network, some of the nodes would behave like Managers while some would be a Peripheral. If we must make the communication process smooth, then the local clocks of these nodes must be synchronized. 

The problem with this synchronization is that we have the clock running in the Manager as well. If we send the value of the Manager clock to the Peripheral, the synchronization doesn’t happen as we have a propagation delay of the messages, along with the propagation delay of the electronic circuits of Manager and the Peripheral.  

The cherry on the cake is that these electronic circuit propagation delays are not random and remain constant, so we can add a time offset to it to match the clock. To tackle this challenge, IEEE has come up with a protocol named “Precision Timing Protocol.” 

Operation of PTP: 

To synchronize the clocks, a Sync message is sent by the Manager to the Peripheral, which then timestamps the receiving time of the same. Following this, a ‘Follow up’ message is issued by the Manager stating the timestamp at which the Sync message was sent. 

The Peripheral then finds the difference between the two values and adds this to its current time. After this, the time difference between the Manager and the Peripheral narrows down to only the propagation delay of the messages.  

To overcome this, the Peripheral issues a ‘Delay Request’ to the Manager, and the Manager, in turn, issues a ‘Delay Response.’ Both these messages have the timestamp of when they were issued. The time at which they are received is then noted. Since two messages are sent, one from the Peripheral and the other from the Manager, there are two propagation delays. Then half of this value is our propagation delay. 

The Peripheral then adds this propagation delay to its clock, and hence the clock gets synchronized. 

Advantages of PTP: 

1. It provides accurate time stamping. 
2. It is a well-known clock synchronization protocol. 
3. It provides intensified security inside the premises. 
4. It provides the possibility of setting coordinated actions and synchronized communication. 

There are various versions of PTP that have been developed over time, namely PTPv1, PTPv2, PTPv2_1, and the latest PTP-AS. 

Cadence Verification IP for Ethernet is available to support the newer version of PTP, allowing simulation of the device for efficient IP, SoC, and system-level design verification. Semiconductor companies can start using it to fully verify their controller design and achieve functional verification closure on it within no time. 

As the title suggests I am unable to perform design sync between OrCAD Capture and Allegro. When I add a layout and try to sync to it I am given ERROR(ORCAP-2426): Cannot run Design Sync because of errors. See session log for error details.

Session Log

[ORPCBFLOW] : Invoking ECO dialog.
INFO(ORNET-1176): Netlisting the design
INFO(ORNET-1178): Design Name:
C:USERSDDOYLEDOCUMENTSCADENCEBOARDSREMOTE POWER DEVICECAPTUREREMOTE_POWER_DEVICE.DSN
Netlist Directory:
c:usersddoyledocumentscadenceoards emote power devicelayoutallegro
Configuration File:
C:CadenceSPB_17.4 ools/capture/allegro.cfg
pstswp.exe - pst - d "C:USERSDDOYLEDOCUMENTSCADENCEBOARDSREMOTE POWER DEVICECAPTUREREMOTE_POWER_DEVICE.DSN"- n "c:usersddoyledocumentscadenceoards emote power devicelayoutallegro" - c "C:CadenceSPB_17.4 ools/capture/allegro.cfg" - v 3 - l 31 - s "" - j "PCB Footprint" - hpath "HPathForCollision"
Spawning... pstswp.exe - pst - d "C:USERSDDOYLEDOCUMENTSCADENCEBOARDSREMOTE POWER DEVICECAPTUREREMOTE_POWER_DEVICE.DSN"- n "c:usersddoyledocumentscadenceoards emote power devicelayoutallegro" - c "C:CadenceSPB_17.4 ools/capture/allegro.cfg" - v 3 - l 31 - s "" - j "PCB Footprint" - hpath "HPathForCollision"
{ Using PSTWRITER 17.4.0 d001Dec-14-2021 at 09:00:49 }

INFO(ORCAP-36080): Scanning netlist files ...

Loading... c:usersddoyledocumentscadenceoards emote power devicelayoutallegropstchip.dat

Loading... c:usersddoyledocumentscadenceoards emote power devicelayoutallegropstchip.dat

Loading... c:usersddoyledocumentscadenceoards emote power devicelayoutallegropstxprt.dat

Loading... c:usersddoyledocumentscadenceoards emote power devicelayoutallegropstxnet.dat
packaging the design view...
Exiting... pstswp.exe - pst - d "C:USERSDDOYLEDOCUMENTSCADENCEBOARDSREMOTE POWER DEVICECAPTUREREMOTE_POWER_DEVICE.DSN"- n "c:usersddoyledocumentscadenceoards emote power devicelayoutallegro" - c "C:CadenceSPB_17.4 ools/capture/allegro.cfg" - v 3 - l 31 - s "" - j "PCB Footprint" - hpath "HPathForCollision"
INFO(ORNET-1179): *** Done ***

This issue started to occur after I changed parts that exist on previously created PCBs. I changed the following leading up to this:

1. Added height in Allegro to many of my components using the Setup->Area->Package Height tool.

2. Changed the reference designator category in OrCAD Capture to TP for several components on board.

Any advice here would be most welcome. Thanks!

Concurrent Layout Editing enables more than one designer to work in a hierarchy at the same time. Check out this blog to know more. (read more)

Training Bytes are not just short technical videos; they are particularly designed to provide comprehensive support in understanding and learning various concepts and methodologies.

These comprehensive yet small Training Bytes can be created to show various concepts and processes in a shorter pane of five to ten minutes, for example, running DFT synthesis, scanning insertion, inserting advanced testability features, test point insertion, debugging DFT violations, etc.

In this blog, we will show you the DFT Synthesis Flow with Cadence's Genus Synthesis Solution using small Training Bytes available on the Cadence Learning and Support Portal. To explore these training bytes more, log on to support.cadence.com and select the learning section to choose the training videos, as shown below.

DFT Synthesis Flow with Genus Synthesis Solution

First, we will understand the Synthesis Flow with DFT in the Genus Synthesis Solution:

Understanding a Script File that Used to Run the Synthesis Flow With DFT

Here, we will show you "How to run the Test Synthesis Flow to Insert Scan Chains and Improve the Testability of a Design" in the Genus Synthesis Solution:

Running Test Synthesis Flow to Insert Scan Chains And Improve the Testability of a Design in the Genus Synthesis Solution

Let's check the flops marked with the dft_mapped attribute for scan mapping in Genus Synthesis Solution:

How to Check Flops Marked With dft_mapped Attribute For Scan Mapping in Genus Synthesis Solution?

How to Find Non-Scan Flops of a Design in Genus? (Video)

Once the flops are mapped to scan flip flops and the scan chain inserted, we will see how to handle the flops marked with the dft_dont_scan attribute for scan mapping in Genus Synthesis Solution.

How to Handle the Flops Marked With the dft_dont_scan Attribute For Scan Mapping in Genus Synthesis Solution?

Here, we will see how to fix DFT Violations using the command fix_dft_violations:

Fixing DFT Violations (Video)

Once the design has been synthesized, let's explore the DFT design hierarchy in Genus Stylus CUI:

Exploring DFT Design Hierarchy in Genus Stylus CUI (Video)

Understand why sequential elements are not mapped to a scan flop:

Why Are Sequential Elements Not Mapped to a Scan Flop?

Explore hierarchical scan synthesis in Genus Stylus Common UI:

Understanding Hierarchical Scan Synthesis in Genus Stylus Common UI. (Video)

To understand how to resolve different warnings and errors (for example, DFT-415, DFT-512, DFT-304, etc.) in Genus Synthesis Solution, here are some videos you can refer to:

How to Resolve Warning: DFT-415 (Video)

How to Resolve Error: DFT-407 (Video)

How to Resolve Error: DFT-404 (Video)

DFT-510 Warning During Mapping (Video)

How to Resolve Warning: DFT-512 (Video)

How to Resolve Warning: DFT-511 (Video)

How to Resolve Warning: DFT-304 (Video)

How to Resolve Warning: DFT-302 (Video)

How to Resolve Error: DFT-515 (Video)

How to Resolve Error: DFT-500 (Video)

Here, we will see how we can generate SDC constraints for DFT constructs for many scan insertion techniques, such as FULLSCAN, OPCG, Boundary Scan, PMBIST, XOR Compression, SmartScan Compression, LBIST, and IEEE 1500:

How to Generate SDC Constraints for DFT Constructs in Genus Synthesis Solution? (Video)

Explore advanced testability features that can be inserted in Genus Synthesis Solution, such as Boundary Scan, Programmable Memory built-in Self-Test Logic (PMBIST), Compression Logic, Masking, and On-Product Clock Generation Logic (OPCG):

Advanced Testability Features (Video)

To understand What the IEEE 1500 Wrapper and its Insertion Flow in Genus Synthesis Solution, follow the bytes:

What Is IEEE 1500 Wrapper? (Video)

IEEE 1500 Wrapper Insertion Flow in Genus Synthesis Solution (Video)

Understand the On-product Clock Generation (OPCG) insertion flow in Genus Synthesis Solution Stylus CUI with this byte:

Understanding On Product Clock Generator (OPCG) Insertion in Genus Stylus CUI (Video)

To debug DFT violations, you can use DFT Analyzer from Genus GUI and explore its features here:

Debugging Using GUI: DFT Analyzer (Video)

Exploring DFT Analyzer View of Genus Synthesis Solution GUI (Video)

To understand What is Shadow Logic, How to Insert Test Points, How to do Testability Analysis Using LBIST, and How to Deterministic Fault Analysis in Genus, follow this article:

What is Shadow Logic

To insert the Boundary Scan Logic in and control Boundary Optimization in Genus Synthesis Solution, refer to these small bytes:

How to Insert Boundary Scan Logic in Genus? Video)

Controlling Boundary Optimization in Genus Synthesis Solution Stylus CUI (Video)

Compression techniques are used during scan insertion to reduce the test data volume and test application time (TAT) while retaining the test coverage. To understand what compression and the compression techniques are, watch this article:

What is Compression Technique During Scan Insertion? (Video)

Interested to know what "Unified Compression" is? To get the concept, you can watch this small demo:

What Is Unified Compression? (Video)

To Explore More, Register for Online Training

• Log on to Cadence.com with your registered Cadence ID and password.
• Select Learning from the menu > Online Courses.
• Search for "Test Synthesis with Genus Stylus Common UI" using the search bar.
• Select the course and click "Enroll."