Functional Role of Dimerization of Human Peptidylarginine Deiminase 4 (PAD4)

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca2+-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (Kd) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The Kd values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower kcat values than that of WT (13.4 s−1). The Kd values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the kcat values of the monomeric mutants ranged from 3.3 to 7.3 s−1. The kcat values of these interface mutants decreased as the Kd values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved]

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work

Antioxidant activity and polyphenols from seaweed and Halimeda Halimeda Opuntia monile

En este trabajo se estudió la actividad antioxidante de dos especies de algas marinas (H. opuntia y H. monile) mediante el ensayo de atrapamiento de radicales DPPH• y el sistema β-Caroteno-acido linoleico. Adicionalmente a las fracciones de ácidos fenolicos libres, ésteres solubles y ésteres insolubles de ácidos fenólicos se les determinó el contenido en fenoles totales mediante la técnica de Folin-Ciocalteu y posteriormente se identificaron y cuantificaron 8 ácidos fenólicos y cinámicos, resultando el componente mayoritario el ácido salicílico. En los ensayos utilizados se obtuvieron valores altos de actividad antioxidante para las diferentes fracciones. A partir de estos resultados se puede postular que la actividad antioxidante de los extractos polares de estas algas pudiera ser explicada, al menos parcialmente, por la presencia de los ácidos fenólicos y cinámicos. En el caso del alga Halimeda monile, de acuerdo con la literatura consultada, es el primer reporte de la actividad antioxidanteIn this paper, the antioxidant activity displayed by two different green seaweed species (H. opuntia y H. monile) was studied using the β- carotene/ linoleic acid and the DPPH• scavenging.systems as different experimental in vitro antioxidant assessment models. Polar seaweed fractions containing free phenolic acids, soluble esters and insoluble esters of phenolic acids were chemically characterized in terms of their phenolic content and composition. In that direction, 8 phenolic acids were identified and quantified, and salycilic acid was shown to be the majoritary compound on the fractions from both species. In addition, the polar fractions were proved to exert antioxidant activity in the two used experimental systems with considerably low values of CI50. Thus, in view of these findings, the antioxidant activity of these polar Halimeda spp. extracts could be supported and at least partially related to the presence of phenolic acids. In case of Halimeda monile this is, at least to the extend of our knowledge, the first report of such biological activity