Logistic regression analysis of populations of electrophysiological models to assess proarrythmic risk.

Population-based computational approaches have been developed in recent years and helped to gain insight into arrhythmia mechanisms, and intra- and inter-patient variability (e.g., in drug responses). Here, we illustrate the use of multivariable logistic regression to analyze the factors that enhance or reduce the susceptibility to cellular arrhythmogenic events. As an example, we generate 1000 model variants by randomly modifying ionic conductances and maximal rates of ion transports in our atrial myocyte model and simulate an arrhythmia-provoking protocol that enhances early afterdepolarization (EAD) proclivity. We then treat EAD occurrence as a categorical, yes or no variable, and perform logistic regression to relate perturbations in model parameters to the presence/absence of EADs. We find that EAD formation is sensitive to the conductance of the voltage-gated Na+, the acetylcholine-sensitive and ultra-rapid K+ channels, and the Na+/Ca2+ exchange current, which matches our mechanistic understanding of the process and preliminary sensitivity analysis. The described technique: •allows investigating the factors underlying dichotomous outcomes, and is therefore a useful tool improve our understanding of arrhythmic risk;•is valid for analyzing both deterministic and stochastic models, and various phenomena (e.g., delayed afterdepolarizations and Ca2+ sparks);•is computationally more efficient than one-at-a-time parameter sensitivity analysis

CaMKII-dependent regulation of cardiac Na(+) homeostasis.

Na(+) homeostasis is a key regulator of cardiac excitation and contraction. The cardiac voltage-gated Na(+) channel, NaV1.5, critically controls cell excitability, and altered channel gating has been implicated in both inherited and acquired arrhythmias. Ca(2) (+)/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine kinase important in cardiac physiology and disease, phosphorylates NaV1.5 at multiple sites within the first intracellular linker loop to regulate channel gating. Although CaMKII sites on the channel have been identified (S516, T594, S571), the relative role of each of these phospho-sites in channel gating properties remains unclear, whereby both loss-of-function (reduced availability) and gain-of-function (late Na(+) current, INa L) effects have been reported. Our review highlights investigating the complex multi-site phospho-regulation of NaV1.5 gating is crucial to understanding the genesis of acquired arrhythmias in heart failure (HF) and CaMKII activated conditions. In addition, the increased Na(+) influx accompanying INa L may also indirectly contribute to arrhythmia by promoting Ca(2) (+) overload. While the precise mechanisms of Na(+) loading during HF remain unclear, and quantitative analyses of the contribution of INa L are lacking, disrupted Na(+) homeostasis is a consistent feature of HF. Computational and experimental observations suggest that both increased diastolic Na(+) influx and action potential prolongation due to systolic INa L contribute to disruption of Ca(2) (+) handling in failing hearts. Furthermore, simulations reveal a synergistic interaction between perturbed Na(+) fluxes and CaMKII, and confirm recent experimental findings of an arrhythmogenic feedback loop, whereby CaMKII activation is at once a cause and a consequence of Na(+) loading

Exploiting the catalytic power of enzymes for oxy- and amino-functionalization reactions

Biocatalysis might effectively address the challenges associated with green and sustainable chemical synthesis. In the work described in this thesis, native and newly engineered enzymes were used for the step-economic and more sustainable biocatalytic synthesis of valuable precursors to pharmaceuticals and nutraceuticals, including enantioenriched epoxides, vicinal triols and amino acids. First, a non-natural aldolase and a non-natural peroxygenase, both evolved in the laboratory from the promiscuous enzyme 4-oxalocrotonate tautomerase, were combined to develop a one-pot two-step enzymatic cascade for the preparation of α,β-epoxy-aldehydes. The addition of a specifically selected natural epoxide hydrolase to the same pot yielded a short pathway to enantioenriched aryl glycerols from simple biomass-derived starting materials. Second, the laboratory evolution of the enzyme ethylenediamine-N,N'-disuccinic acid (EDDS) lyase generated an enzyme variant that showed a large improvement in activity, enabling the effective and enantioselective synthesis of complex L-aspartic acid derivatives, valuable precursors to the artificial dipeptide sweeteners neotame and advantame. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives. Finally, a study into the structure, mechanism and biocatalytic potential of a newly discovered monooxygenase, named Rhop3, is presented

X-ray study of a sample of FR0 radio galaxies: unveiling the nature of the central engine

FR0s are compact radio sources that represent the bulk of the Radio-Loud (RL) AGN population, but they are still poorly understood. Pilot studies on these sources have been already performed at radio and optical wavelengths: here we present the first X-ray study of a sample of 19 FR0 radio galaxies selected from the SDSS/NVSS/FIRST sample of Best & Heckman (2012), with redshift $\leq$ 0.15, radio size $\leq$ 10 kpc and optically classified as low-excitation galaxies (LEG). The X-ray spectra are modeled with a power-law component absorbed by Galactic column density with, in some cases, a contribution from thermal extended gas. The X-ray photons are likely produced by the jet as attested by the observed correlation between X-ray (2-10 keV) and radio (5 GHz) luminosities, similar to FRIs. The estimated Eddington-scaled luminosities indicate a low accretion rate. Overall, we find that the X-ray properties of FR0s are indistinguishable from those of FRIs, thus adding another similarity between AGN associated with compact and extended radio sources. A comparison between FR0s and low luminosity BL Lacs, rules out important beaming effects in the X-ray emission of the compact radio galaxies. FR0s have different X-ray properties with respect to young radio sources (e.g. GPS/CSS sources), generally characterized by higher X-ray luminosities and more complex spectra. In conclusion, the paucity of extended radio emission in FR0s is probably related to the intrinsic properties of their jets that prevent the formation of extended structures, and/or to intermittent activity of their engines.Comment: Accepted for publication in MNRAS (18 pages, 4 figures

The gamma-ray emission region in the FRII Radio Galaxy 3C 111

The Broad Line Radio Galaxy 3C 111, characterized by a Fanaroff-Riley II (FRII) radio morphology, is one of the sources of the Misaligned Active Galactic Nuclei sample, consisting of Radio Galaxies and Steep Spectrum Radio Quasars, recently detected by the Fermi-Large Area Telescope. Our analysis of the 24-month gamma-ray light curve shows that 3C 111 was only occasionally detected at high energies. It was bright at the end of 2008 and faint, below the Fermi-Large Area Telescope sensitivity threshold, for the rest of the time. A multifrequency campaign of 3C~111, ongoing in the same period, revealed an increase of the mm, optical and X-ray fluxes in 2008 September-November, interpreted by Chatterjee et al. (2011) as due to the passage of a superluminal knot through the jet core. The temporal coincidence of the mm-optical-X-ray outburst with the GeV activity suggests a co-spatiality of the events, allowing, for the first time, the localization of the gamma-ray dissipative zone in a FRII jet. We argue that the GeV photons of 3C 111 are produced in a compact region confined within 0.1 pc and at a distance of about 0.3 pc from the black hole.Comment: 8 pages, 4 figures. ApJL in pres

Identification, comprehensive characterization, and comparative genomics of the HERV-K(HML8) integrations in the human genome

Around 8% of the human genome is composed by Human Endogenous Retroviruses (HERVs), ancient viral sequences inherited from the primate germ line after their infection by now extinct retroviruses. Given the still underexplored physiological and pathological roles of HERVs, it is fundamental to increase our information about the genomic composition of the different groups, to lay reliable foundation for functional studies. Among HERVs, the most characterized elements belong to the beta-like superfamily HERV-K, comprising 10 groups (HML1-10) with HML2 being the most recent and studied one. Among HMLs, the HML8 group is the only one still lacking a comprehensive genomic description. In the present work, we investigated HML8 sequences' distribution in the human genome (GRCh38/hg38), identifying 23 novel proviruses and characterizing the overall 78 HML8 proviruses in terms of genome structure, phylogeny, and integration pattern. HML8 elements were significantly enriched in human chromosomes 8 and X (p<0.005) while chromosomes 17 and 20 showed fewer integrations than expected (p<0.025 and p<0.005, respectively). Phylogenetic analyses classified HML8 members into 3 clusters, corresponding to the three LTR types MER11A, MER11B and MER11C. Besides different LTR types, common signatures in the internal structure suggested the potential existence of three different ancestral HML8 variants. Accordingly, time of integration estimation coupled with comparative genomics revealed that these three clusters have a different time of integration in the primates' genome, with MER11C elements being significantly younger than MER11A- and MER11B associated proviruses (p<0.005 and p<0.05, respectively). Approximately 30% of the HML8 elements were found co-localized within human genes, sometimes in exonic portions and with the same orientation, deserving further studies for their possible effects on gene expression. Overall, we provide the first detailed picture of the HML8 group distribution and variety among the genome, creating the backbone for the specific analysis of their transcriptional activity in healthy and diseased conditions

Enhancing the Peroxygenase Activity of a Cofactor‐Independent Peroxyzyme by Directed Evolution Enabling Gram‐Scale Epoxide Synthesis

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H(2)O(2) with water being the sole by‐product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4‐oxalocrotonate tautomerase (4‐OT), catalysing the epoxidation of a variety of α,β‐unsaturated aldehydes with H(2)O(2), is outstanding because of its independence from any cost‐intensive cofactor. However, its low‐level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β‐epoxy‐aldehydes under preparative‐scale conditions is limiting the potential of 4‐OT. Herein we report the directed evolution of a tandem‐fused 4‐OT variant, which showed an ∼150‐fold enhanced peroxygenase activity compared to 4‐OT wild type, enabling the synthesis of α,β‐epoxy‐aldehydes in milligram‐ and gram‐scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor‐independent peroxyzyme can provide new opportunities for the eco‐friendly and practical synthesis of enantioenriched epoxides at large scale