Rapid detection of nanoplastics and small microplastics by Nile-Red staining and flow cytometry

Microplastics are of rising health concerns because they have been detected even in remote and pristine environments, from the Artic snow to the Marianne Trench. The occurrence and impact of nanoplastics in ecosystems is almost unknown, in particular due to analytical limitations such as very small sizes that fall below detection limits of current techniques. Here we take advantage of a common interference in analytical flow cytometry to develop a method for the quantification of the number of plastic particles in the 0.6-15 mu m size range. Plastic particles are stained with the lipophilic dye Nile-Red then detected by flow cytometry, a method regularly used in biology for rapid quantification of fluorescent cells. We found that sample analysis lasts 90 s, which is hundreds of times faster than the analysis of filter portions by micro-Raman and other spectroscopic techniques. Our method is highly efficient in detecting polyethylene, with staining efficiency higher than 70% and signal linearity with concentration. Staining efficiency up to 96% was observed for polyvinylchloride and for polystyrene.Peer reviewe

Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

<p>Abstract</p> <p>Background</p> <p>Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins.</p> <p>Results</p> <p>Metazoan PBP-βL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-βL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-βL homologs comprise four alloparalogus protein lineages that derive from α-proteobacteria.</p> <p>Conclusion</p> <p>While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-βL proteins were conserved and are found in metazoans including humans. Metazoan PBP-βL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-βL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding.</p

LACTB is a filament-forming protein localized in mitochondria

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function

Amino acid alignment of catalytic site motifs and gene architecture of LACTB orthologs

() Schematic organization and alignment of the three PBP-βLs catalytic site motifs (highlighted in green) and their flanking regions in LACTB orthologs. The corresponding motifs in the strain R61 D-alanyl-D-alanine carboxypeptidase [Swiss-Prot:] are included. Abbreviations for species names, in order: Homsa, (Swiss-Prot:); Macmu, (Ensembl:ENSMMUP00000004719); Musmu, (Swiss-Prot:); Orycu, (Ensembl:ENSOCUP00000008608); Canfa, (RefSeq:XP_544713); Bosta, (Swiss-Prot:); Mondo, (Ensembl:ENSMODP00000013893); Galga,(Swiss-Prot:); Xentr, (Ensembl:ENSXETG00000009720); Fugru, (Ensembl:SINFRUP00000138119); Oryla, (Ensembl:ENSORLP00000009787); Gasac, (Ensembl:ENSGACP00000014046); Danre, (RefSeq:NP_001018429); Strpu, (RefSeq:XP_789736); Cioin, (Ensembl:ENSCING00000006798); Schja, (GenPept:AAX27853, AAX25200); Caeel, (RefSeq:NP_001041033); Caebr, (GenPept:CAE74593); Dicdi, (Swiss-Prot:); Stesp, strain R61. The mitochondrial import sequence () is indicated. Amino acid conserved in all taxa are highlighted in yellow. () Organization of exons and introns in LACTB genes of representative metazoan taxa.<p><b>Copyright information:</b></p><p>Taken from "Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy"</p><p>http://www.biomedcentral.com/1471-2148/8/26</p><p>BMC Evolutionary Biology 2008;8():26-26.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266909.</p><p></p

Schematic representation of the organization of the three catalytic site motifs in LACTB and the different PBP-βL classes

A set of founding members of each PBP-βL class (Additional file ), classified according to Ghuysen 1997, and Massova and Mobashery 1998 [3,4], were used to calculate the median inter-motif distances in number of amino acid residues. Accession numbers refer to the Swiss-Prot database. The catalytic site motifs are highlighted in green and invariant amino acids are higlighted in yellow. Inter-motif distances were measured from the serine in the -SXXK-motif to the serine/lysine and lysine/histidine of the second and third catalytic site motif, respectively. Numbers within brackets is the largest difference from the median value within each class. PBP-βL classes forming separate clades [3] are marked with square brackets. Abbreviations: PBP, penicillin-binding protein.<p><b>Copyright information:</b></p><p>Taken from "Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy"</p><p>http://www.biomedcentral.com/1471-2148/8/26</p><p>BMC Evolutionary Biology 2008;8():26-26.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266909.</p><p></p