Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5′ Terminal Regions

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5′ terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5′ TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5′ TR of JEV and demonstrated its direct interaction with recombinant DDX3X (K_d of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5′ and 3′ TRs of flaviviruses, we investigated if the ZIKV 5′ TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5′ TR with a K_d of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5′ TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication     

Chemical Synthesis and Biological Applications of O-GlcNAcylated Peptides and Proteins

All human cells use O-GlcNAc protein modifications (O-linked N-acetylglucosamine) to rapidly adapt to changing nutrient and stress conditions through signaling, epigenetic, and proteostasis mechanisms. A key challenge for biologists in defining precise roles for specific O-GlcNAc sites is synthetic access to homogenous isoforms of O-GlcNAc proteins, a result of the non-genetically templated, transient, and heterogeneous nature of O-GlcNAc modifications. Toward a solution, this review details the state of the art of two strategies for O-GlcNAc protein modification: advances in “bottom-up” O-GlcNAc peptide synthesis and direct “top-down” installation of O-GlcNAc on full proteins. We also describe key applications of synthetic O-GlcNAc peptide and protein tools as therapeutics, biophysical structure–function studies, biomarkers, and as disease mechanistic probes to advance translational O-GlcNAc biology.     

Predicting and interpreting identification errors in military vehicle training using multidimensional scaling

We compared methods for predicting and understanding the source of confusion errors during military vehicle identification training. Participants completed training to identify main battle tanks. They also completed card-sorting and similarity-rating tasks to express their mental representation of resemblance across the set of training items. We expected participants to selectively attend to a subset of vehicle features during these tasks, and we hypothesised that we could predict identification confusion errors based on the outcomes of the card-sort and similarity-rating tasks. Based on card-sorting results, we were able to predict about 45% of observed identification confusions. Based on multidimensional scaling of the similarity-rating data, we could predict more than 80% of identification confusions. These methods also enabled us to infer the dimensions receiving significant attention from each participant. This understanding of mental representation may be crucial in creating personalised training that directs attention to features that are critical for accurate identification.

Practitioner Summary: Participants completed military vehicle identification training and testing, along with card-sorting and similarity-rating tasks. The data enabled us to predict up to 84% of identification confusion errors and to understand the mental representation underlying these errors. These methods have potential to improve training and reduce identification errors leading to fratricide.     

Synthesis of novel tri- and tetrasubstituted C<Subscript>18</Subscript> furan fatty esters

A methylene-interrupted C₁₈ keto-acetylenic fatty ester (methyl 12-oxo-9-octadecynoate) was obtained from methyl ricinoleate by bromination-dehydrobromination followed by oxidation. Reaction of methyl 12-oxo-9-octadecynoate with bis(benzonitrile) palladium(II) chloride, allyl bromide, or methyl-allyl bromide furnished methyl 8-[5-hexyl-3-allyl-furan-2-yl]-octanoate (1, 56%) or methyl 8-[5-hexyl-3-(2-methyl-allyl)-furan-2-yl]-octanoate (2, 55%). Reaction of methyl 12-oxo-11-chloro-or 11-fluoro-9-octadeyynoate (prepared from methyl santalbate-methyl 11-E-9-octadecynoate, found in sandalwood, Santalum album, seed oil) with bis(benzonitrile) palladium(II) chloride gave methyl 8-(4-fluoro-5-hexyl-furan-2-yl)-octanoate (3, 50%) or methyl 8-(4-fluoro-5-hexyl-furan-2-yl)-octanoate (4, 50%), respectively. And when methyl 12-oxo-11-chloro- or 11-fluoro-9-octadecynoate was treated with a mixture of bis(benzonitrile) palladium(II) chloride, allyl bromide, or methyl-allyl bromide, the reaction yielded tetrasubstituted C₁₈ furan derivatives, viz, methyl 8-(3-allyl-4-chloro-5-hexyl-furan-2-yl)-octanoate (5, 54%), methyl 8-[4-chloro-5-hexyl-3-(2-methyl-allyl)-furan-2-yl)-octanoate (6, 54%), methyl 8-(3-allyl-4-fluoro-5-hexyl-furan-2-yl]-octanoate (7, 10%), and methyl 8-[4-fluoro-5-hexyl-3-(2-methyl-allyl)-furan-2-yl]-octanoate (8, 10%). The presence of a fluorine atom in the furan derivatives 4, 7, and 8 was readily characterized by the appearance of doublets for carbon nuclei, which were coupled to the fluorine atom in the ¹³C NMR spectra. All furan fatty derivatives from this work were characterized by NMR spectroscopic and mass spectrometric analyses. The yields of compounds 7 and 8 were very low (10%) despite attempts to improve the procedure by increasing the amounts of the reactants and catalyst.     

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues

Pactamycin is a bacteria‐derived aminocyclitol antibiotic with a wide‐range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.     

Using yeast RNA as a probe for generation of hydroxyl radicals by earth materials

Inhalation of certain types of particulate matter can lead to lung disease. The reactivity of these particles and, in part, the pathologic responses that result are dictated by their physicochemical properties. The ability of particles to induce the generation of reactive oxygen species (ROS), especially hydroxyl radicals in vivo, is one property that has been correlated to the development of lung disease. Several minerals, such as quartz and asbestos, are known to generate hydroxyl radicals and cause lung disease, but many other minerals have never been tested. Here, we describe a technique employing yeast RNA as a probe to screen for mineral-generated hydroxyl radicals. The stability of RNA in the presence of hydrogen peroxide, ferrous iron, hydroxyl radicals, and several common minerals (quartz, albite, forsterite, fayalite, hematite, magnetite, coal, and pyrite) was examined. 3'-(p-Aminophenyl) fluorescein (APF) was used to verify mineral generation of ROS. RNA is stable in the presence of hydrogen peroxide, quartz, and albite; while it degrades in the presence of ferrous iron, hydroxyl radicals, and the other minerals. Coal and pyrite are the most reactive both in RNA degradation and hydroxyl radical generation. This noncellular technique provides a straightforward way to compare many different particles simultaneously. Those particles showing reactivity toward RNA using this method are high-priority candidates for further in vitro and possibly in vivo tests.     

Optimization of Co-content in C14 Laves phase multi-component alloys for NiMH battery application

The structural, electrochemical, and gas phase hydrogen storage properties of predominantly C14 crystalline phase alloys with partial replacement by Co (up to 2.5 at%) are reported. Minor phases, including C15 and cubic TiNi, were found by X-ray diffraction and contributed to the improved hydrogen diffusion through the alloy. The optimal Co-content of between 1.0 and 1.5 at% provides easy activation, high gas phase capacity, and a high discharge capacity. An optimized alloy formula also provided the smallest metallic nickel cluster size embedded in the surface oxide and the largest number of nickel clusters. In sealed cell studies, a Co-content of 1.5% gives the best performance in formation, cycle life, and charge retention, but has worse specific power and low temperature performance than some other compositions. The high-rate dischargeabilities at lower rates were found to be limited by surface reactions, not bulk diffusion.     

The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion

The classical action of the hormone 1,25-dihydroxyvitamin D3 (VD) is the regulation of calcium metabolism. In contrast, the peptide hormone atrial natriuretic factor (ANF) is one of the few known nonclassical VD responding genes. We screened the promoter of the rat ANF gene and identified a typical VD receptor (VDR) binding site formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides, between positions -907 and -891. Like most of the DR3-type VD response elements this sequence is bound with high affinity (Kd = 0.53 nM) by a heterodimer formed by VDR and retinoid X receptor. In a heterologous promoter context one copy of this sequence mediated an about fourfold gene activation by VD and a half-maximal activation (EC50) value of 0.48 nM VD. This characterizes the identified sequence as one of the most potent VD response elements.