ResearchPad - chemical-reactions https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study]]> https://www.researchpad.co/article/elastic_article_13877 Type 1 diabetes is a risk factor for coronary heart disease. The underlying mechanism behind the accelerated atherosclerosis formation is not fully understood but may be related to the formation of oxidation products and advanced glycation end-products (AGEs). We aimed to examine the associations between the collagen oxidation product methionine sulfoxide; the collagen AGEs methylglyoxal hydroimidazolone (MG-H1), glucosepane, pentosidine, glucuronidine/LW-1; and serum receptors for AGE (RAGE) with measures of coronary artery disease in patients with long-term type 1 diabetes.MethodsIn this cross-sectional study, 99 participants with type 1 diabetes of ≥ 45-year duration and 63 controls without diabetes had either established coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA) measuring total, calcified and soft/mixed plaque volume. Skin collagen methionine sulfoxide and AGEs were measured by liquid chromatography-mass spectrometry and serum sRAGE/esRAGE by ELISA.ResultsIn the diabetes group, low levels of methionine sulfoxide (adjusted for age, sex and mean HbA1c) were associated with normal coronary arteries, OR 0.48 (95% CI 0.27–0.88). Glucuronidine/LW-1 was associated with established CHD, OR 2.0 (1.16–3.49). MG-H1 and glucuronidine/LW-1 correlated with calcified plaque volume (r = 0.23–0.28, p<0.05), while pentosidine correlated with soft/mixed plaque volume (r = 0.29, p = 0.008), also in the adjusted analysis.ConclusionsLow levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes, suggesting a role for metabolic and oxidative stress in the formation of atherosclerosis in diabetes. ]]> <![CDATA[Evaluation of residue management practices on barley residue decomposition]]> https://www.researchpad.co/article/elastic_article_13875 Optimizing barley (hordeum vulgare L.) production in Idaho and other parts of the Pacific Northwest (PNW) should focus on farm resource management. The effect of post-harvest residue management on barley residue decomposition has not been adequately studied. Thus, the objective of this study was to determine the effect of residue placement (surface vs. incorporated), residue size (chopped vs. ground-sieved) and soil type (sand and sandy loam) on barley residue decomposition. A 50-day(d) laboratory incubation experiment was conducted at a temperature of 25°C at the Aberdeen Research and Extension Center, Aberdeen, Idaho, USA. Following the study, a Markov-Chain Monte Carlo (MCMC) modeling approach was applied to investigate the first-order decay kinetics of barley residue. An accelerated initial flush of residue carbon(C)-mineralization was measured for the sieved (Day 1) compared to chopped (Day 3 to 5) residues for both surface incorporated applications. The highest evolution of carbon dioxide (CO2)-C of 8.3 g kg-1 dry residue was observed on Day 1 from the incorporated-sieved application for both soils. The highest and lowest amount of cumulative CO2-C released and percentage residue decomposed over 50-d was observed for surface-chopped (107 g kg-1 dry residue and 27%, respectively) and incorporated-sieved (69 g kg-1 dry residue and 18%, respectively) residues, respectively. There were no significant differences in C-mineralization from barley residue based on soil type or its interactions with residue placement and size (p >0.05). The largest decay constant k of 0.0083 d-1 was calculated for surface-chopped residue where the predicted half-life was 80 d, which did not differ from surface sieved or incorporated chopped. In contrast, incorporated-sieved treatments only resulted in a k of 0.0054 d-1 and would need an additional 48 d to decompose 50% of the residue. Future residue decomposition studies under field conditions are warranted to verify the residue C-mineralization and its impact on residue management.

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<![CDATA[Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance]]> https://www.researchpad.co/article/elastic_article_7690 We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

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<![CDATA[Concept of an artificial muscle design on polypyrrole nanofiber scaffolds]]> https://www.researchpad.co/article/elastic_article_8464 Here we present the synthesis and characterization of two new conducting materials having a high electro-chemo-mechanical activity for possible applications as artificial muscles or soft smart actuators in biomimetic structures. Glucose-gelatin nanofiber scaffolds (CFS) were coated with polypyrrole (PPy) first by chemical polymerization followed by electrochemical polymerization doped with dodecylbenzensulfonate (DBS-) forming CFS-PPy/DBS films, or with trifluoromethanesulfonate (CF3SO3-, TF) giving CFS-PPy/TF films. The composition, electronic and ionic conductivity of the materials were determined using different techniques. The electro-chemo-mechanical characterization of the films was carried out by cyclic voltammetry and square wave potential steps in bis(trifluoromethane)sulfonimide lithium solutions of propylene carbonate (LiTFSI-PC). Linear actuation of the CFS-PPy/DBS material exhibited 20% of strain variation with a stress of 0.14 MPa, rather similar to skeletal muscles. After 1000 cycles, the creeping effect was as low as 0,2% having a good long-term stability showing a strain variation per cycle of -1.8% (after 1000 cycles). Those material properties are excellent for future technological applications as artificial muscles, batteries, smart membranes, and so on.

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<![CDATA[Impact of confinement in vehicle trunks on decomposition and entomological colonization of carcasses]]> https://www.researchpad.co/article/Nffbdbe54-85a9-48b9-9e05-57433aec6303

In order to investigate the impact of confinement in a car trunk on decomposition and insect colonization of carcasses, three freshly killed pig (Sus scrofa domesticus Erxleben) carcasses were placed individually in the trunks of older model cars and deployed in a forested area in the southwestern region of British Columbia, Canada, together with three freshly killed carcasses which were exposed in insect-accessible protective cages in the same forest. Decomposition rate and insect colonization of all carcasses were examined twice a week for four weeks. The exposed carcasses were colonized immediately by Calliphora latifrons Hough and Calliphora vomitoria (L.) followed by Lucilia illustris (Meigen), Phormia regina (Meigen) and Protophormia terraenovae (R.-D.) (Diptera: Calliphoridae). There was a delay of three to six days before the confined carcasses were colonized, first by P. regina, followed by Pr. terraenovae. These species represented the vast majority of blow fly species on the confined carcasses. Despite the delay in colonization, decomposition progressed much more rapidly in two of the confined carcasses in comparison with the exposed carcasses due to the greatly increased temperatures inside the vehicles, with the complete skeletonization of two of the confined carcasses ocurring between nine and 13 days after death. One confined carcass was an anomaly, attracting much fewer insects, supporting fewer larval calliphorids and decomposing much more slowly than other carcasses, despite similarly increased temperatures. It was later discovered that the vehicle in which this carcass was confined had a solid metal fire wall between the passenger area and the trunk, which served to reduce insect access and release of odors. These data may be extremely valuable when analyzing cadavers found inside vehicle trunks.

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<![CDATA[Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway]]> https://www.researchpad.co/article/5c784fbdd5eed0c484007497

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

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<![CDATA[SimKinet: A free educational tool based on an electrical analogy to solve chemical kinetic equations]]> https://www.researchpad.co/article/5c8c1982d5eed0c484b4d822

In this article we introduce the software SimKinet, a free tool specifically designed to solve systems of differential equations without any programming skill. The underlying method is the so-called Network Simulation Method, which designs and solves an electrical network equivalent to the mathematical problem. SimKinet is versatile, fast, presenting a real user-friendly interface, and can be employed for both educational and researching purposes. It is particularly useful in the first courses of different scientific degrees, mainly Chemistry and Physics, especially when facing non-analytic or complex-dynamics problems. Moreover, SimKinet would help students to understand fundamental concepts, being an opportunity to improve instruction in Chemistry, Mathematics, Physics and other Sciences courses, with no need of advanced knowledge in differential equations. The potency of SimKinet is demonstrated via two applications in chemical kinetics: the photochemical destruction of stratospheric ozone and the chaotic dynamics of the peroxidase-oxidase reaction.

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<![CDATA[Probabilistic analysis of a concrete column in an aggressive soil environment]]> https://www.researchpad.co/article/5c8acce9d5eed0c4849902cb

Sulphate attack is one of the most important factors that limit the lifetime of pure concrete constructions. Harsh environmental conditions have a large impact on the operational costs of concrete columns or piles dipped into soil. The results are non-deterministic; therefore, reliability analysis is often used. The strength characteristics of the substrate around the construction were modelled as one-dimensional prismatic beams related with random p-y curves. Sulphate deterioration is defined as a set of random variables jointed with two dimensional mechanical systems at acceptable levels. Fick’s second law describes the penetration of sulphate ingress into pure concrete with explicit numerical solutions for boundary conditions and an increase in the transition factor under the progress of sulphate ingress. This process was partially solved via analytical methods for sulphate ion transport and numerically for a random field. This solves the mechanical task and determines the system reliability. A numerical example is provided to illustrate the proposed method to prevent unexpected structural failures during column service life. The proposed methodology can assist designers and can help to make decisions on existing foundations to ensure the safety of geotechnical construction.

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<![CDATA[Genome-wide DNA methylation analysis of pituitaries during the initiation of puberty in gilts]]> https://www.researchpad.co/article/5c8accc9d5eed0c48498ffd8

It has been widely recognized that the early or delayed puberty appears to display harmful effects on adult health outcomes. During the timing of puberty, pituitaries responds to the hypothalamus and then introduce the following response of ovaries in hypothalamic-pituitary-gonadal axis. DNA methylation has been recently suggested to regulate the onset of puberty in female mammals. However, to date, the changes of DNA methylation in pituitaries have not been investigated during pubertal transition. In this study, using gilts as the pubertal model, the genome-scale DNA methylation of pituitaries was profiled and compared across Pre-, In- and Post-puberty by using the reduced representation bisulfite sequencing. We found that average methylation levels of each genomic feature in Post- were lower than Pre- and In-pubertal stage in CpG context, but they were higher in In- than that in Pre- and Post-pubertal stage in CpH (where H = A, T, or C) context. The methylation patterns of CpHs were more dynamic than that of CpGs at the location of high CpG content, low CpG content promoter genes, and differently genomic CGIs. Furthermore, the differently genomic CGIs were likely to show in a similar manner in CpG context but display in a stage-specific manner in the CpH context across the Pre-, In- and Post-pubertal stage. Among these pubertal stages, 5 kb upstream regions of the transcription start sites were protected from both CpG and CpH methylation changes. 12.65% of detected CpGs were identified as the differentially methylated CpGs, regarding 4301 genes which were involved in the fundamental functions of pituitaries. 0.35% of detected CpHs were identified as differentially methylated CpHs, regarding 3691 genes which were involved in the biological functions of releasing gonadotropin hormones. These observations and analyses would provide valuable insights into epigenetic mechanism of the initiation of puberty in pituitary level.

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<![CDATA[Molecular mechanisms of mesoporous silica formation from colloid solution: Ripening-reactions arrest hollow network structures]]> https://www.researchpad.co/article/5c9902e3d5eed0c484b9881a

The agglomeration of silica nanoparticles in aqueous solution is investigated from molecular simulations. Mimicking destabilization of colloidal solutions by full removal of protective moieties or surface charge, association of SiO2/Si(OH)4 core/shell particles leads to rapid proton transfer reactions that account for local silanole → silica ripening reactions. Yet, such virtually barrier-less binding is only observed within a limited contact zone. Agglomeration hence leads to the formation of oligomers of nanoparticles, whilst full merging into a compact precipitate is hampered by the need for extended structural reorganisation. Implementing sufficiently fast supply from colloidal solution, our simulations show the development of silica networks comprised of covalently bound, yet not fully merged nanoparticles. Within the oligomerized nanoparticle network, coordination numbers range from 2 to 5 –which is far below closest packing. Our simulations hence rationalize the formation of covalently bound network structures hosting extended pores. The resulting interfaces to the solvent show water immobilization only for the immediate contact layers, whilst the inner pores exhibit solvent mobility akin to bulk water.

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<![CDATA[Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin]]> https://www.researchpad.co/article/5c818e96d5eed0c484cc260a

The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.

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<![CDATA[Effects of increased space allowance on animal welfare, meat and ham quality of heavy pigs slaughtered at 160Kg]]> https://www.researchpad.co/article/5c706779d5eed0c4847c70a2

Sixty barrows (Body Weight–BW- range: 23.9–160 kg) were allotted to two experimental groups (6 pens of 5 pigs each): the control group was kept at a space allowance of 1m2/head; the second group was kept at 1.3m2/head. Behaviour, growth parameters, carcass and meat quality were assessed, as well as fat and cured ham quality. Results showed that pigs raised at 1.3m2/head spent more time laying (particularly in lateral recumbency, P<0.01 and P<0.001, respectively) compared to pigs kept at lower space allowance. They also reduced the aimless exploration of the slatted pen floor (P<0.001) and increased overall expression of other, mainly active, behaviors (e.g., drinking, walking and standing, P<0.01). Pigs raised at 1.3m2/head showed higher final BW (P = 0.02), more favourable Average Daily Gain (ADG) and gain-to-Feed ratio (G:F) both during the last period of the trial (P<0.05 for both parameters) and over the entire trial (P = 0.01 for both parameters). No significant difference was observed between groups for carcass traits and the main meat quality attributes. Subcutaneous fat from green hams had higher α-linolenic acid content (P<0.01) in the group reared at greater space allowance. Green hams from this group lost less weight at trimming (P<0.01) and the resulting cured hams received better sensory evaluations (P<0.05). No difference was observed in fatty acid composition and unsaturation levels of the subcutaneous fat from cured hams. Our data suggest that heavy pigs intended for Parma ham would benefit from the adoption of higher individual floor space allowances, both in terms of animal welfare (increased possibility to rest) and of productive parameters, without having any detrimental effect on the suitability of the thighs for dry-curing or on the quality of the final product.

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<![CDATA[Epigenome-wide analysis of sperm cells identifies IL22 as a possible germ line risk locus for psoriatic arthritis]]> https://www.researchpad.co/article/5c75ac57d5eed0c484d085ff

Psoriasis and its associated inflammatory arthritis, psoriatic arthritis (PsA), have a clear heritable component, but a large proportion of the heritable risk remains unexplained by gene sequence variation. This study aimed to determine if epigenetic factors contribute to the missing heritability in psoriatic disease. DNA methylation profiling was performed on sperm cells from 23 probands with psoriasis without PsA (PsC), 13 PsA probands, and 18 unaffected controls. Differentially methylated CpGs and regions (DMRs) were identified and validated by pyrosequencing. Underlying AluY and copy number variation (CNV) in the HCG26 and IL22 genes, respectively, were assessed by genotyping. Array, subject’s age, age of psoriasis onset, psoriasis severity, and medication usage were found to influence methylation at many genes and were included as covariates in the analysis. Between PsC probands vs. controls, 169 DMRs were found; 754 DMRs were found between PsA probands vs. controls, and 86 between PsA and PsC probands (adjusted p<0.05). Differences in methylation across DMRs were generally subtle (<10%) but correlated well with pyrosequencing. Biological inference prioritized notable DMRs associated with skin disease (SIGLEC14, JAM3, PCOLCE, RXRB), skin and/or joint disease (MBP, OSBPL5, SNORD115, HCG26), and joint disease (IL22, ELF5, PPP2R2D, PTPRN2, HCG26). Hypermethylation of the DMR within the first exon of arthritis-associated IL22 showed significant correlation (rho = 0.34, 95% CI 0.06–0.57, p = 0.01) between paired sperm and blood samples, independent of a CNV within the same region. Further studies are needed to rule out underlying genetic causes and determine if these represent heritable, constitutional epimutations, or are the result of exposure of germ cells to endogenous or exogenous environmental factors.

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<![CDATA[An African-specific haplotype in MRGPRX4 is associated with menthol cigarette smoking]]> https://www.researchpad.co/article/5c706741d5eed0c4847c6cc6

In the U.S., more than 80% of African-American smokers use mentholated cigarettes, compared to less than 30% of Caucasian smokers. The reasons for these differences are not well understood. To determine if genetic variation contributes to mentholated cigarette smoking, we performed an exome-wide association analysis in a multiethnic population-based sample from Dallas, TX (N = 561). Findings were replicated in an independent cohort of African Americans from Washington, DC (N = 741). We identified a haplotype of MRGPRX4 (composed of rs7102322[G], encoding N245S, and rs61733596[G], T43T), that was associated with a 5-to-8 fold increase in the odds of menthol cigarette smoking. The variants are present solely in persons of African ancestry. Functional studies indicated that the variant G protein-coupled receptor encoded by MRGPRX4 displays reduced agonism in both arrestin-based and G protein-based assays, and alteration of agonism by menthol. These data indicate that genetic variation in MRGPRX4 contributes to inter-individual and inter-ethnic differences in the preference for mentholated cigarettes, and that the existence of genetic factors predisposing vulnerable populations to mentholated cigarette smoking can inform tobacco control and public health policies.

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<![CDATA[Temporal evolution and pathway models of poly(ethylene-terephthalate) degradation under multi-factor accelerated weathering exposures]]> https://www.researchpad.co/article/5c70673ad5eed0c4847c6c71

Photolytic and hydrolytic degradation of poly(ethylene-terephthalate) (PET) polymers with different stabilizers were performed under multiple accelerated weathering exposures and changes in the polymers were monitored by various evaluation techniques. Yellowing was caused by photolytic degradation and haze formation was induced by combined effects of photolytic and hydrolytic degradation. The formation of light absorbing chromophores and bleaching of the UV stabilizer additive were recorded through optical spectroscopy. Chain scission and crystallization were found to be common mechanisms under both photolytic and hydrolytic conditions, based on the infrared absorption of the carbonyl (C = O) band and the trans ethylene glycol unit, respectively. The degradation mechanisms determined from these evaluations were then used to construct a set of degradation pathway network models using the network structural equation modeling (netSEM) approach. This method captured the temporal evolution of degradation by assessing statistically significant relationships between applied stressors, mechanistic variables, and performance level responses. Quantitative pathway equations provided the contributions from mechanistic variables to the response changes.

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<![CDATA[Fat cells gobbling up norepinephrine?]]> https://www.researchpad.co/article/5c65dcd9d5eed0c484dec37d

The sympathetic nervous system (SNS) controls key aspects of adipose tissue (AT) function through the release of norepinephrine (NE) and beta adrenergic signaling. Sympathetic tone is determined by NE release but also by the rate of extracellular NE clearance that historically has been believed to occur solely through solute carrier family 6 member 2 (SLC6A2) expressed on sympathetic neurons. Song and colleagues show that adipocytes can also clear NE through organic cation transporter 3 (Oct3). This contributes to our understanding of how adrenergic signaling is controlled in AT and also emphasizes the need to develop better methods to assess adrenergic signaling in vivo.

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<![CDATA[Reproductive characteristics modify the association between global DNA methylation and breast cancer risk in a population-based sample of women]]> https://www.researchpad.co/article/5c6f1484d5eed0c48467a21a

DNA methylation has been implicated in breast cancer aetiology, but little is known about whether reproductive history and DNA methylation interact to influence carcinogenesis. This study examined modification of the association between global DNA methylation and breast cancer risk by reproductive characteristics. A population-based case-control study assessed reproductive history in an interviewer-administered questionnaire. Global DNA methylation was measured from white blood cell DNA using luminometric methylation assay (LUMA) and pyrosequencing assay (long interspersed elements-1 (LINE-1). We estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) among 1 070 breast cancer cases and 1 110 population-based controls. Effect modification was assessed on additive and multiplicative scales. LUMA methylation was associated with elevated breast cancer risk across all strata (comparing the highest to the lowest quartile), but estimates were higher among women with age at menarche ≤12 years (OR = 2.87, 95%CI = 1.96–4.21) compared to >12 years (OR = 1.66, 95%CI = 1.20–2.29). We observed a 2-fold increase in the LUMA methylation-breast cancer association among women with age at first birth >23 years (OR = 2.62, 95%CI = 1.90–3.62) versus ≤23 years (OR = 1.32, 95% CI = 0.84–2.05). No modification was evident for parity or lactation. Age at menarche and age at first birth may be modifiers of the association between global DNA methylation and breast cancer risk.

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<![CDATA[Synthesis of MnCo2O4 nanoparticles as modifiers for simultaneous determination of Pb(II) and Cd(II)]]> https://www.researchpad.co/article/5c648ce7d5eed0c484c81a65

The porous spinel oxide nanoparticles, MnCo2O4, were synthesized by citrate gel combustion technique. Morphology, crystallinity and Co/Mn content of modified electrode was characterized and determined by Fourier transform infra-red spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), X-ray diffraction pattern analysis (XRD), simultaneous thermogravimetry and differential thermal analysis (TG/DTA). Nanoparticles were used for modification of glassy carbon electrode (GCE) and new sensor was applied for simultaneous determination of Pb(II) and Cd(II) ions in water samples with the linear sweep anodic stripping voltammetry (LSASV).The factors such as pH, deposition potential and deposition time are optimized. Under optimal conditions the wide linear concentration range from 0.05 to 40 μmol/dm3was obtained for Pb(II), with limit of detection (LOD) of 8.06 nmol/dm3 and two linear concentration ranges were obtained for Cd(II), from 0.05 to 1.6 μmol/dm3 and from 1.6 to 40 μmol/dm3, with calculated LOD of 7.02 nmol/dm3. The selectivity of the new sensor was investigated in the presence of interfering ions. The sensor is stable and it gave reproducible results. The new sensor was succesfully applied on determination of heavy metals in natural waters.

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<![CDATA[Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM]]> https://www.researchpad.co/article/5c50c481d5eed0c4845e8843

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

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<![CDATA[Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds]]> https://www.researchpad.co/article/5c5df370d5eed0c4845812dc

Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2’s catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG) and maximal rate constant (k’ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of ‘kinetic’ disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.

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