Multiplex Genomic Tagging of Mammalian ATG8s to Study Autophagy.

Atg8 proteins play a crucial role in autophagy. There is a single Atg8 isoform in yeast, while mammals have up to seven homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16, implicated in various stages along the pathway. However, the intricacies among GABARAP proteins are complex and require a more precise delineation. Here, we introduce a new cellular platform to study autophagy using CRISPR/Cas9-mediated tagging of endogenous genes of the GABARAP subfamily with different fluorescent proteins. This platform allows robust examination of autophagy by flow cytometry of cell populations and monitoring of GABARAP homologs at single-cell resolution using fluorescence microscopy. Strikingly, the simultaneous labeling of the different endogenous GABARAPs allows the identification and isolation of autophagosomes differentially marked by these proteins. Using this system, we found that the different GABARAPs are associated with different autophagosomes. We argue that this new cellular platform will be crucial in studying the unique roles of individual GABARAP proteins in autophagy and other putative cellular processes.

Utilization of diverse organophosphorus pollutants by marine bacteria.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

The Meat Quality Characteristics of Holstein Calves: The Story of Israeli 'Dairy Beef'.

Global animal production systems are often criticized for their lack of sustainability and insufficient resilience to ensure food security. The 'farm-to-fork' approach aims at orienting food systems towards the creation of a positive environmental impact, nutritious, healthy, safe and sufficient foods, and fairer economic returns for primary producers. Many countries rely on an imported supply of live animals to fulfill their needs for fresh meat. In Israel, ~60% of the sources of fresh beef come from the import of live animals. In order to encourage sustainable beef production in Israel, the proportion of local beef should be raised at the expense of imported animals. However, for this to be achieved, the superior performance of local beef should be justified. The current study was conducted to compare between the meat quality characteristics of local (Israeli Holstein; N = 205) vs. imported (Australian; N = 169) animals. Generally, while the imported calves presented a higher dressing percentage (p < 0.0001), the local animals were characterized by tenderer meat (p < 0.0001), longer sarcomeres (p < 0.0001), higher a* color attributes and pH (p < 0.001), superior cooking (p = 0.002) and thawing loss (p < 0.0001), higher intra-muscular fat (IMF) content, and a higher PUFA proportion (p < 0.01 and p < 0.0001, respectively) and PUFA:SFA ratio. The findings shown herein may provide sound arguments for stakeholders and policy makers to facilitate sustainable local beef production in Israel.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.