Coordination of KSHV Latent and Lytic Gene Control by CTCF-Cohesin Mediated Chromosome Conformation

Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5′ and 3′ end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control

Mobilization of pro-inflammatory lipids in obese Plscr3-deficient mice

Metabolic profiling of mice deficient in phospholipid scramblase 3 reveals a possible molecular link between obesity and inflammation

Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid

The excited-state structure, vibrations, lifetimes, and nonradiative dynamics of jet-cooled 1-methylcytosine

We have investigated the S0 → S1 UV vibronic spectrum and time-resolved S1 state dynamics of jet-cooled amino-keto 1-methylcytosine (1MCyt) using two-color resonant two-photon ionization, UV/UV holeburning and depletion spectroscopies, as well as nanosecond and picosecond timeresolved pump/delayed ionization measurements. The experimental study is complemented with spin-component-scaled second-order coupled-cluster and multistate complete active space second order perturbation ab initio calculations. Above the weak electronic origin of 1MCyt at 31 852 cm−1 about 20 intense vibronic bands are observed. These are interpreted as methyl group torsional transitions coupled to out-of-plane ring vibrations, in agreement with the methyl group rotation and out-of-plane distortions upon 1ππ∗ excitation predicted by the calculations. The methyl torsion and ν′1 (butterfly) vibrations are strongly coupled, in the S1 state. The S0 → S1 vibronic spectrum breaks off at a vibrational excess energy Eexc ∼ 500 cm−1, indicating that a barrier in front of the ethylene-type S1 S0 conical intersection is exceeded, which is calculated to lie at Eexc = 366 cm−1. The S1 S0 internal conversion rate constant increases from kIC = 2 · 109 s−1 near the S1(v = 0) level to 1 · 1011 s−1 at Eexc = 516 cm−1. The 1ππ∗ state of 1MCyt also relaxes into the lower-lying triplet T1 (3ππ∗) state by intersystem crossing (ISC); the calculated spin-orbit coupling (SOC) value is 2.4 cm−1. The ISC rate constant is 10–100 times lower than kIC; it increases from kISC = 2 · 108 s−1 near S1(v = 0) to kISC = 2 · 109 s−1 at Eexc = 516 cm−1. The T1 state energy is determined from the onset of the time-delayed photoionization efficiency curve as 25 600 ± 500 cm−1. The T2 (3nπ∗) state lies >1500 cm−1 above S1(v = 0), so S1 T2 ISC cannot occur, despite the large SOC parameter of 10.6 cm−1. An upper limit to the adiabatic ionization energy of 1MCyt is determined as 8.41 ± 0.02 eV. Compared to cytosine, methyl substitution at N1 lowers the adiabatic ionization energy by ≥0.32 eV and leads to a much higher density of vibronic bands in the S0 → S1 spectrum. The effect of methylation on the radiationless decay to S0 and ISC to T1 is small, as shown by the similar break-off of the spectrum and the similar computed mechanismsThis research has been supported by the Schweiz. Nationalfonds (Grant Nos. 121993 and 132540), the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from Catalonia (Spain) (Grant No. 2014SGR1202), the Ministerio de Economía y Competividad (MINECO) from Spain (Grant No. CTQ2015-69363-P), and the National Natural Science Foundation of China (Grant No. 21303007

Role of Medium- and Short-Chain L-3-Hydroxyacyl-CoA Dehydrogenase in the Regulation of Body Weight and Thermogenesis

Dysregulation of fatty acid oxidation plays a pivotal role in the pathophysiology of obesity and insulin resistance. Medium- and short-chain-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (SCHAD) (gene name, hadh) catalyze the third reaction of the mitochondrial β-oxidation cascade, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, for medium- and short-chain fatty acids. We identified hadh as a putative obesity gene by comparison of two genome-wide scans, a quantitative trait locus analysis previously performed in the polygenic obese New Zealand obese mouse and an earlier described small interfering RNA-mediated mutagenesis in Caenorhabditis elegans. In the present study, we show that mice lacking SCHAD (hadh(−/−)) displayed a lower body weight and a reduced fat mass in comparison with hadh(+/+) mice under high-fat diet conditions, presumably due to an impaired fuel efficiency, the loss of acylcarnitines via the urine, and increased body temperature. Food intake, total energy expenditure, and locomotor activity were not altered in knockout mice. Hadh(−/−) mice exhibited normal fat tolerance at 20 C. However, during cold exposure, knockout mice were unable to clear triglycerides from the plasma and to maintain their normal body temperature, indicating that SCHAD plays an important role in adaptive thermogenesis. Blood glucose concentrations in the fasted and postprandial state were significantly lower in hadh(−/−) mice, whereas insulin levels were elevated. Accordingly, insulin secretion in response to glucose and glucose plus palmitate was elevated in isolated islets of knockout mice. Therefore, our data indicate that SCHAD is involved in thermogenesis, in the maintenance of body weight, and in the regulation of nutrient-stimulated insulin secretion

Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets

The activation of complement during platelet activation is incompletely understood.We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets.C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation.C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

Peer reviewe

Interactions between selected bile salts and Triton X-100 or sodium lauryl ether sulfate

<p>Abstract</p> <p>Background</p> <p>In order to develop colloidal drug carriers with desired properties, it is important to determine physico-chemical characteristics of these systems. Bile salt mixed micelles are extensively studied as novel drug delivery systems. The objective of the present investigation is to develop and characterize mixed micelles of nonionic (Triton X-100) or anionic (sodium lauryl ether sulfate) surfactant having oxyethylene groups in the polar head and following bile salts: cholate, deoxycholate and 7-oxodeoxycholate.</p> <p>Results</p> <p>The micellization behaviour of binary anionic-nonionic and anionic-anionic surfactant mixtures was investigated by conductivity and surface tension measurements. The results of the study have been analyzed using Clint's, Rubingh's, and Motomura's theories for mixed binary systems. The negative values of the interaction parameter indicate synergism between micelle building units. It was noticed that Triton X-100 and sodium lauryl ether sulfate generate the weakest synergistic interactions with sodium deoxycholate, while 7-oxodeoxycholate creates the strongest attractive interaction with investigated co-surfactants.</p> <p>Conclusion</p> <p>It was concluded that increased synergistic interactions can be attributed to the larger number of hydrophilic groups at α side of the bile salts. Additionally, 7-oxo group of 7-oxodeoxycholate enhance attractive interactions with selected co-surfactants more than 7-hydroxyl group of sodium cholate.</p

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson's disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread