Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Phosphorylation of the voltage-gated Na⁺ (Na_V) channel Na_V1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed Na_V1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on Na_V1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic Na_V1.5 mutants revealed the roles of three phosphosites in regulating Na_V1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface Na_V1.5 expression and peak Na⁺ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular Na_V1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of Na_V1.5 channels is highly complex, site specific, and dynamic.     

Hepatitis C virus infection may lead to slower emergence of P. falciparum in blood

Background

Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV) and hepatitis C virus (HCV) overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria.

Methodology

We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction.

Principal Findings

At inclusion, 65 (20.4%) subjects had detectable malaria parasites in blood, 36 (11.3%) were HBV chronic carriers, and 61 (18.9%) were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection.

Conclusions

This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens, which could help in identifying new therapeutic approaches against malaria.     

Control of early events in olfactory processing by adult neurogenesis

The mature brain needs to have flexible control over behavior in the face of ever-changing needs. It achieves this control through morphological and physiological changes at the level of molecules, spines, dendrites, and axons and through processes of adult neurogenesis, entire cells. The functional maturation of newly generated cells in the adult forebrain involves the expression of neurotransmitter receptors before synaptic activity and excitatory gamma-aminobutyric acid (GABAergic) influences prior to glutamatergic input. The production of new cells for incorporation into neural circuits that are already up and running gives rise to a unique situation that may require epigenetic regulation. However, once mature, new neurons must carve out a niche among more established cells to be useful. How do they survive and what are they used for? Recent studies have revealed that adult neurogenesis alters the olfactory bulb at all levels, from single cells to the network and system levels. It has also been suggested that cell turnover may be particularly beneficial for the processing of new information in dynamic networks. However, elucidating the functional meaning of adult neurogenesis must wait for the development of new paradigms to eliminate the pool of newly generated neurons but sparing the preexisting ones. Nevertheless, there is already considerable correlative evidence to indicate that adult neurogenesis is a plastic mechanism by which the performance of the brain can be optimized in a given environment.     

Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands

EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of Eth through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new surface plasmon resonance (SPR) methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose-dependent negative SPR signal. We demonstrate that this signal reveals the affinity of small molecules for the repressor. The affinity constants (K_D) correlate with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes in EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result opens perspectives onto the development of an SPR assay that would at the same time reveal structural changes in the target upon binding with an inhibitor and the binding constant of this interaction.     

Association of Residual Plasma Viremia and Intima-Media Thickness in Antiretroviral-Treated Patients with Controlled Human Immunodeficiency Virus Infection

Background

While residual plasma viremia is commonly observed in HIV-infected patients undergoing antiretroviral treatment (ART), little is known about its subclinical consequences.

Methods

This cross-sectional study included 47 male, never-smoking, non-diabetic patients with ≥4 years of ART and controlled HIV-replication (HIV-viral load, VL <20 copies/mL for ≥1 year). Residual HIV-VL was measured using an ultrasensitive assay (quantification limit: 1 copy/ml). Patients were categorized as having detectable (D; 1-20 copies/mL, n = 14) or undetectable (UD; <1 copies/mL, n = 33) HIV-VL. Linear regression was used to model the difference in total carotid intima-media thickness [c-IMT, measures averaged across common carotid artery (cca), bifurcation, and internal carotid artery] and cca-IMT alone across detection groups. Multivariable models were constructed for each endpoint in a forward-stepwise approach.

Results

No significant differences were observed between viremia groups with respect to median ART-duration (9.6 years, IQR = 6.8–10.9), nadir CD4+T-cell (208/mm³, IQR = 143–378), and CD4+T-cell count (555/mm³, IQR = 458–707). Median adjusted inflammatory markers tended to be higher in patients with D- than UD-viremia, with differences in IL-10 being significant (p = 0.03). After adjustment on age, systolic blood pressure, and insulin resistance, mean cca-IMT was significantly lower in patients with undetectable (0.668 mm±0.010) versus detectable viremia (0.727 mm±0.015, p = 0.002). Cca-IMT was also independently associated with age and insulin resistance. Mean adjusted total c-IMT was no different between viremia groups (p = 0.2), however there was large variability in bifurcation c-IMT measurements.

Conclusions

Higher cca-IMT was observed in patients with detectable, compared to undetectable, HIV-VL in never-smoking ART-controlled patients, suggesting that residual HIV viremia may be linked to atherosclerosis.     

Assays for transfer RNA-dependent amino acid biosynthesis

Selenocysteinyl-tRNA(Sec), cysteinyl-tRNA(Cys), glutaminyl-tRNA(Gln), and asparaginyl-tRNA(Asn) in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the tRNA. This non-cognate amino acid is then converted to the cognate amino acid by a tRNA-dependent modifying enzyme. The in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-tRNA, is labile and requires a prior enzymatic step to be synthesized. The need to separate product aa-tRNA from unreacted substrate is typically a labor- and time-intensive task; this adds another impediment in the investigation of these enzymes. Here, we review four different approaches for studying these tRNA-dependent amino acid modifications. In addition, we describe in detail a [32P]/nuclease P1 assay for glutaminyl-tRNA(Gln) and asparaginyl-tRNA(Asn) formation which is sensitive, enables monitoring of the aminoacyl state of the tRNA, and is less time consuming than some of the other techniques. This [32P]/nuclease P1 method should be adaptable to studying tRNA-dependent selenocysteine and cysteine synthesis.     

Cationized starch-based material as a new ion-exchanger adsorbent for the removal of C.I. Acid Blue 25 from aqueous solutions

This article describes the use of a cationized starch-based material as new ion-exchanger adsorbent for the removal of C.I. Acid Blue 25 (AB 25) from aqueous solutions. Batch adsorption studies concerning the effects of contact time, pH and temperature are presented and discussed. Adsorption experimental data showed that: (i) the process was uniform and rapid: adsorption of dye reached equilibrium in 50 min in the wide pH range of dye solutions; (ii) adsorption kinetics followed the pseudo-second order model; (iii) the Langmuir model yielded a much better fit than the Freundlich model for the dye concentration range under study; (iv) this adsorbent exhibited interesting adsorption capacities: on the basis of the Langmuir analysis, the maximum adsorption capacity was determined to be 322 mg of dye per gram of material at 25 degrees C; (v) the adsorption capacity decreased with increasing temperature; and (vi) the negative value of free energy change indicated the spontaneous nature of adsorption.     

Life cycle assessment of a steam thermolysis process to recover carbon fibers from carbon fiber-reinforced polymer waste

Purpose

Carbon fibers have been widely used in composite materials, such as carbon fiber-reinforced polymer (CFRP). Therefore, a considerable amount of CFRP waste has been generated. Different recycling technologies have been proposed to treat the CFRP waste and recover carbon fibers for reuse in other applications. This study aims to perform a life cycle assessment (LCA) to evaluate the environmental impacts of recycling carbon fibers from CFRP waste by steam thermolysis, which is a recycling process developed in France.

Methods

The LCA is performed by comparing a scenario where the CFRP waste is recycled by steam-thermolysis with other where the CFRP waste is directly disposed in landfill and incineration. The functional unit set for this study is 2 kg of composite. The inventory analysis is established for the different phases of the two scenarios considered in the study, such as the manufacturing phase, the recycling phase, and the end-of-life phase. The input and output flows associated with each elementary process are standardized to the functional unit. The life cycle impact assessment (LCIA) is performed using the SimaPro software and the Ecoinvent 3 database by the implementation of the CML-IA baseline LCIA method and the ILCD 2011 midpoint LCIA method.

Results and discussion

Despite that the addition of recycling phase produces non-negligible environmental impacts, the impact assessment shows that, overall, the scenario with recycling is less impactful on the environment than the scenario without recycling. The recycling of CFRP waste reduces between 25 and 30% of the impacts and requires about 25% less energy. The two LCIA methods used, CML-IA baseline and ILCD 2011 midpoint, lead to similar results, allowing the verification of the robustness and reliability of the LCIA results.

Conclusions

The recycling of composite materials with recovery of carbon fibers brings evident advantages from an environmental point of view. Although this study presents some limitations, the LCA conducted allows the evaluation of potential environmental impacts of steam thermolysis recycling process in comparison with a scenario where the composites are directly sent to final disposal. The proposed approach can be scaled up to be used in other life cycle assessments, such as in industrial scales, and furthermore to compare the steam thermolysis to other recycling processes.