Conradsenmacmillan2353

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the ratios of enriched extracts in the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modular. Our work thus serves as an effective proof-of-concept for a scheme of "metabolic biosensing", which should enable rapid, field-deployable detection of complex organic water contaminants.Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response ( less then 10 μM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of less then 1 μM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.In this work, the 3-D structure of the well-known opioid drug heroin in a solution was investigated. The goal was to provide a complete and detailed description of the stable conformers with their relative abundances. This knowledge is very important from the pharmaceutical and forensic point of view as it could help significantly with deeper understanding of heroin's metabolism and the development of antagonist medicines for the case of an overdose. As heroin is a chiral compound with five stereogenic centres, the methods of chiroptical spectroscopy supplemented by density functional theory (DFT) calculations were applied to study its conformations in chloroform solution. The selected chiroptical methods, namely, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD), are inherently sensitive to the 3-D structure of small- to medium-sized chiral organic molecules. A thorough conformational analysis revealed four stable conformers of heroin in chloroform solution, where the conductor-like polarizable continuum model of the solvent was used for all the calculations. The simulated ultraviolet (UV), infrared (IR), ECD, and VCD spectra were compared with the experimental ones and very good agreement was found, which enabled a detailed structure description and interpretation of the spectra. Chiroptical spectroscopy in combination with DFT calculations proved to be a very sensitive tool for the analysis of the 3-D structure of heroin in a solution in contrast with conventional spectroscopic methods. Especially, the application of VCD seems to be a promising approach for monitoring structural changes, for instance, those caused by solvents or interactions with other agents. this website © 2020 Wiley Periodicals, Inc.Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV-OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV-CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium-mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA-B in ToLCNDV-OM.