The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-A resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.

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Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs.

The stability of solutions of the antitumour antimetabolites, vinca alkaloids, podophyllotoxins, interferons, steroids and platinum drugs as well as maytansine, asparaginase, amsacrine, flavone-8-acetic acid, mitoguazone, and N-phosphonoacetyl-L-aspartate (PALA) is reviewed. Much of the published work has been done with biological, not stability-indicating, assays; thus, the relevant results should be used with caution. With this proviso, almost all of these drugs can be stored in solution for several days at room temperature or 4°C. Most reports also suggest that the drugs that have been tested are stable when frozen in solution. For a number of the drugs, particular precautions are required; for instance, amsacrine should not be mixed with chloride-containing solutions, whereas cisplatin is most stable in solutions containing >0.1 M chloride.

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. II. Assay methods, adriamycin and the other antitumour antibiotics.

Niche nanoparticle-based FRET assay for bleomycin detection via DNA scission.

Bleomycins (BLMs) are widely used in combination with chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and the potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLM content in both pharmaceutical analysis and clinical samples, to make full use of therapeutic efficacy and to weaken its toxicity. Here, we proposed a simple, rapid, and convenient electrochemical assay for trace BLM detection. A reported DNA motif, as substrate for BLMs, is prepared to self-assemble onto the gold electrode to fabricate an electrochemical DNA (E-DNA) sensor, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as a signal reporter to form a stem-loop structure, giving an arise of remarkable faradaic current. In the presence of Fe(II)·BLM, t...

Sensitive DNA-Based Electrochemical Strategy for Trace Bleomycin Detection

High-Performance Liquid Chromatographic Determination of Components of Bleomycin Preparations

The mercuric acetate method (Method I) cited in the Code of Federal Regulations (CFR) as the official method for determining the penicillamine content of a drug was compared with a nonaqueous amine titration (Method II), a nonaqueous acid titration (Method III), and a hydroxylamine assay (Method IV). Quantitative determinations were made for 3 types of penicillamine samples: commercial lots of bulk drug, capsules, and test solutions containing known amounts of penicillamine and EDTA. Method III proved unsatisfactory in the bulk drug determinations. Method II, although slightly less precise than Method I, gave generally satisfactory results for the bulk drug and capsule samples. Method IV was the only method to give satisfactory results in the presence of EDTA.

Comparison of methods for determining penicillamine in bulk drug and formulations.

Infrared Determination of Tetramethylthiuram Disulfide in Commercial Dithiocarbamate Formulations by the Potassium Bromide Disk Technique

Several methods for quantitating probenecid were compared: USP XIX method for probenecid in tablets, a modified USP extraction method for probenecid in tablets, a new column extraction method for probenecid in tablets and oral suspensions containing ampicillin, and a high pressure liquid chromatographic (HPLC) method for probenecid in tablets and oral suspensions containing antibiotics. The first 3 methods were satisfactory for probenecid in bulks and tablets but proved unsatisfactory for oral suspeonsions containing flavors. The flavors gave positive interference for probenecid in oral suspensions. The HPLC method, although more time-consuming, separated probenecid from excipients, thus eliminating positive interference from flavors.

Comparison of methods for determining probenecid in tablets and flavored oral suspensions containing ampicillin.

A high pressure liquid chromatographic method for determining probenecid in oral suspensions of amoxicillin was applied to the determination of probenecid in oral suspensions of ampicillin. Three preparations containing various known amounts of probenecid in synthetic mixtures of ampicillin oral suspensions were analyzed by 5 chemists in an intralaboratory study, with satisfactory results. Blind duplicates of 3 prepared oral suspensions were sent to 12 collaborators, who were instructed to analyze the samples in a fixed random order. The standards showed a satisfactory linear response. Average recoveries of probenecid in the interlaboratory study for the 6 mixtures ranged from 95.2 to 99.1%, and the coefficients of variation ranged from 1.63 to 4.9%.

Paul J Vollmer

Papers

High-Performance Liquid Chromatographic Determination of Components of Bleomycin Preparations

Comparison of methods for determining penicillamine in bulk drug and formulations.

Infrared Determination of Tetramethylthiuram Disulfide in Commercial Dithiocarbamate Formulations by the Potassium Bromide Disk Technique

Comparison of methods for determining probenecid in tablets and flavored oral suspensions containing ampicillin.

High pressure liquid chromatographic method for probenecid in flavored oral suspensions containing ampicillin: collaborative studies.