Engineered nanoparticles are increasingly incorporated into consumer products and are emerging as potential environmental contaminants. Upon environmental release, nanoparticles could inhibit bacterial processes, as evidenced by laboratory studies. Less is known regarding bacterial alteration of nanoparticles, including whether bacteria affect physical agglomeration states controlling nanoparticle settling and bioavailability. Here, the effects of an environmental strain of Pseudomonas aeruginosa on TiO 2 nanoparticle agglomerates formed in aqueous media are described. Environmental scanning electron microscopy and cryogenic scanning electron microscopy visually demonstrated bacterial dispersion of large agglomerates formed in cell culture medium and in marsh water. For experiments in cell culture medium, quantitative image analysis verified that the degrees of conversion of large agglomerates into small nanoparticle-cell combinations were similar for 12-h-growth and short-term cell contact experiments. Dispersion in cell growth medium was further characterized by size fractionation: for agglomerated TiO 2 suspensions in the absence of cells, 81% by mass was retained on a 5-m-pore-size filter, compared to only 24% retained for biotic treatments. Filtrate cell and agglomerate sizes were characterized by dynamic light scattering, revealing that the average bacterial cell size increased from 1.4 m to 1.9 m because of nano-TiO 2 biosorption. High-magnification scanning electron micrographs showed that P. aeruginosa dispersed TiO 2 agglomerates by preferential biosorption of nanoparticles onto cell surfaces. These results suggest a novel role for bacteria in the environmental transport of engineered nanoparticles, i.e., growth-independent, bacterially mediated size and mass alterations of TiO 2 nanoparticle agglomerates.

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acylCoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V max of 8.7 nmol/min/mg protein and an apparent K m of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn 21 ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [ 3 H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked.This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.-Stålberg, K., A. C. Neal, H. Ronne, and U. Ståhl. Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae.

SummaryLong-chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for virtually every metabolic pathway that catabolizes FA or synthesizes complex lipids. We have hypothesized that each of the five cloned ACSL isoforms partitions FA towards specific downstream pathways. Adult heart expresses all five cloned ACSL isoforms, but their independent functional roles have not been elucidated. Studies implicate ACSL1 in both oxidative and lipid synthetic pathways. To clarify the functional role of ACSL1 and the other ACSL isoforms (3-6), we examined ACS specific activity and Acsl mRNA expression in the developing mouse heart which increases FA oxidative pathways for energy production after birth. Compared to the embryonic heart, ACS specific activity was 14-fold higher on post-natal day 1 (P1). On P1, as compared to the fetus, only Acsl1 mRNA increased, whereas transcripts for the other Acsl isoforms remained the same, suggesting that ACSL1 is the major isoform responsible for activating long-chain FA for myocardial oxidation after birth. In contrast, the mRNA abundance of Acsl3 was highest on E16, and decreased dramatically by P7, suggesting that ACSL3 may play a critical role during the development of the fetal heart. Our data support the hypothesis that each ACSL has a specific role in the channeling of FA towards distinct metabolic fates. Keywordsfatty acid activation; heart ontogeny; heart fatty acid oxidation; heart lipid synthesis; heart phospholipid composition Abbreviations ACO, acyl-CoA oxidase; ACS, acyl-CoA synthetase; ACSL and Acsl, long chain acyl-CoA synthetase protein and mRNA, respectively; CPT-1, carnitine palmitoyl transferase-1; DTT, dithiothreitol; E16 embryonic day 16; FA, fatty acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LCAD, long-chain acyl dehydrogenase; MCAD, medium-chain acyl dehydrogenase; P1, postnatal day 1; PPAR, peroxisome proliferator-activated receptor

Background: Some patients with neovascular age-related macular degeneration (nAMD) have persistent intraretinal/subretinal fluid (IRF/SRF) despite being treated with anti-VEGF agents. There is limited data on efficacy of switching to intravitreal brolucizumab (IVBr) in these patients. Purpose: To determine anatomic and visual outcomes of eyes with nAMD treated with for persistent IRF/SRF. Methods: Retrospective series of eyes with nAMD treated initially with aflibercept (IVA, n = 48) and bevacizumab (IVBe, n = 10), then switched to IVBr for persistent IRF/SRF. Results: In the IVA-IVBr group, a mean of 42 days after one IVBr, mean logMAR changed from 0.50 to 0.49 ( p = 0.73) and mean CSFT changed from 340 to 305 µm ( p < 0.001); 31% of eyes had no fluid, 42% had persistent but reduced fluid, 25% had stable fluid, and 2% had increased fluid. For a subgroup of 25 eyes that completed a series of 3 IVBr, mean logMAR changed from 0.44 to 0.40 ( p = 0.35) and mean CSFT changed from 325 to 277 µm ( p = 0.001); 24% of eyes had no fluid at last follow-up, a mean of 54 days after last IVBr. In the IVBe-IVBr group, a mean of 44 days after one IVBr, mean logMAR changed from 0.46 to 0.40 ( p = 0.114) and mean CSFT from 401 to 325 µm ( p = 0.009); 30% of eyes had no fluid and 70% had persistent but reduced fluid. For a subgroup of four eyes that completed a series of three IVBr, mean logMAR changed from 0.33 to 0.35 ( p = 0.391) and mean CSFT improved from 375 to 275 µm ( p = 0.001); 50% of eyes had no fluid at last follow-up, a mean of 65 days after last IVBr. Conclusion: In nAMD eyes previously treated with IVA and IVBe, switching to IVBr significantly reduced persistent IRF/SRF but did not significantly affect visual outcomes.

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

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