The miRNA pathway has three segments—biogenesis, targeting and downstream regulatory effectors. We aimed to better understand their cellular control by exploring the miRNA-mRNA-targeting relationships. We first used human evolutionarily conserved sites. Strikingly, AGOs 1–3 are all among the top 14 mRNAs with the highest miRNA site counts, along with ANKRD52, the phosphatase regulatory subunit of the recently identified AGO phosphorylation cycle; and the AGO phosphorylation cycle mRNAs share much more than expected miRNA sites. The mRNAs for TNRC6, which acts with AGOs to channel miRNA-mediated regulatory actions onto specific mRNAs, are also heavily miRNA-targeted. In contrast, upstream miRNA biogenesis mRNAs are not, and neither are downstream regulatory effectors. In short, binding site enrichment in miRNA targeting machinery mRNAs, but neither upstream biogenesis nor downstream effector mRNAs, was observed, endowing a cellular capacity for intensive and specific feedback control of the targeting activity. The pattern was confirmed with experimentally determined miRNA-mRNA target relationships. Moreover, genetic experiments demonstrated cellular utilization of this capacity. Thus, we uncovered a capacity for intensive, and specific, feedback-regulation of miRNA targeting activity directly by miRNAs themselves, i.e. segment-specific feedback auto-regulation of miRNA pathway, complementing miRNAs pairing with transcription factors to form hybrid feedback-loop.

During the COVID-19 pandemic, the increase in demand for protective equipment caused a global shortage and homemade barrier face coverings were recommended as alternatives. However, filtration performances of homemade face coverings have not been fully evaluated. Test methods in the ASTM standard (F3502-21) were used to evaluate filtration efficiencies (FE) and breathability (pressure drop, Δp) of face coverings and home fabric materials commonly used during the pandemic. Submicron particulates FE was measured by particle transmission through face covering samples using a Condensation Particle Counter equipped with differential mobility analyzer and electronic manometer. Flow resistance of 0.1 μm-diameter fluorescent nanoparticles in droplets was determined by measuring fluorescence intensity of residual collected at the reverse side of samples. The size-dependent FE (3–94%) and Δ p (0.8–72 mmH 2 O) varied considerably among fabrics. Of the 16 mask types, 31.25% and 81.25% met the minimum FE and breathability standards in the ASTM F3502-21, respectively. Overall performance (qF) was highest for velcro masks (max qF = 3.36, min qF = 2.80) and lowest for Dutch wax print fabrics (max qF = 0.12, min qF = 0.03). Most of the samples resisted the flow of 0.1 µm-diameter nanoparticles in droplets. Low flow resistance was observed in bandana, neck gaiter, t-shirt I, tank top and bedspread fabrics. GSM and fabric finishing seems to affect performance. Low performances can be improved by selecting optimum-performance fabrics in the design and manufacture of barrier face coverings. Supplementary Information The online version contains supplementary material available at 10.1007/s41742-021-00390-6.

AbstractMany experimental protocols have to be carried out in vitro due to a lack of cell-permeable analysis probes. For instance, the cellular signaling moderator RAS proteins alternate between the active GTP-binding and the inactive GDP-binding states. Though many GTP analogs can serve as probes for RAS activity analysis, their cell impermeability renders in vivo analysis impossible. On the other hand, the lipid/calcium/phosphate (LCP) nanoparticle has enabled efficient intracellular delivery of a nucleotide analog as a chemotherapy agent. Thus, using RAS analysis and LCP nanoparticle as the prototype, we tackled the cell-impermeability issue via nanoparticle-mediated intracellular delivery of the analysis probe. Briefly, BODIPY-FT-GTP-γ-S, a GTP analog that becomes fluorescent only upon protein binding, was chosen as the analysis probe, so that GTP binding can be quantified by fluorescent activity. BODIPY-FT-GTP-γ-S-loaded LCP-nanoparticle was synthesized for efficient intracellular BODIPY-FT-GTP-γ-S delivery. Binding of the delivered BODIPY-FT-GTP-γ-S to the RAS proteins were consistent with previously reported observations; the RAS GTP binding activity was reduced in serum-starved cells; and a transient activation peak of the binding activity was observed upon subsequent serum reactivation of the cells. In a word, nanoparticle-mediated probe delivery enabled an in vivo RAS analysis method. The approach should be applicable to a wide variety of analysis protocols.

The human kinome contains >500 protein kinases, and regulates up to 30% of all human proteins. Kinase study is currently hindered by a lack of in vivo analysis approaches, mainly due to two factors: our inability to distinguish the kinase reaction of interest from those of other members of the kinome in live cells and the cell impermeability of the ATP analogs. Herein, we aimed to overcome this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the Shokat method, was used as our initial prototype. Briefly, following the Shokat method, enlargement of the ATP binding pocket was performed by mutating the gate-keeper Methionine residue to a Glycine, prompting the mutant AKT1 to preferentially use the bulky ATP analog N 6 -Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The LPC nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. We demonstrated that the mutant, but not the wild type, AKT1 was able to use the delivered A*TPγS for autophosphorylation as well as phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.