The active zone protein Clarinet regulates synaptic sorting of ATG-9 and presynaptic autophagy.

Autophagy is essential for cellular homeostasis and function. In neurons, autophagosome biogenesis is temporally and spatially regulated to occur near presynaptic sites, in part via the trafficking of autophagy transmembrane protein ATG-9. The molecules that regulate autophagy by sorting ATG-9 at synapses remain largely unknown. Here, we conduct forward genetic screens at single synapses of C. elegans neurons and identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating sorting of autophagy protein ATG-9 at synapses, and presynaptic autophagy. We determine that disrupting CLA-1L results in abnormal accumulation of ATG-9 containing vesicles enriched with clathrin. The ATG-9 phenotype in cla-1(L) mutants is not observed for other synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Through genetic analyses, we uncover the adaptor protein complexes that genetically interact with CLA-1 in ATG-9 sorting. We also determine that CLA-1L extends from the active zone to the periactive zone and genetically interacts with periactive zone proteins in ATG-9 sorting. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic autophagy.

Comparison of head direction cell firing characteristics across thalamo-parahippocampal circuitry.

Head direction (HD) cells, which fire persistently when an animal's head is pointed in a particular direction, are widely thought to underlie an animal's sense of spatial orientation and have been identified in several limbic brain regions. Robust HD cell firing is observed throughout the thalamo-parahippocampal system, although recent studies report that parahippocampal HD cells exhibit distinct firing properties, including conjunctive aspects with other spatial parameters, which suggest they play a specialized role in spatial processing. Few studies, however, have quantified these apparent differences. Here, we performed a comparative assessment of HD cell firing characteristics across the anterior dorsal thalamus (ADN), postsubiculum (PoS), parasubiculum (PaS), medial entorhinal (MEC), and postrhinal (POR) cortices. We report that HD cells with a high degree of directional specificity were observed in all five brain regions, but ADN HD cells display greater sharpness and stability in their preferred directions, and greater anticipation of future headings compared to parahippocampal regions. Additional analysis indicated that POR HD cells were more coarsely modulated by other spatial parameters compared to PoS, PaS, and MEC. Finally, our analyses indicated that the sharpness of HD tuning decreased as a function of laminar position and conjunctive coding within the PoS, PaS, and MEC, with cells in the superficial layers along with conjunctive firing properties showing less robust directional tuning. The results are discussed in relation to theories of functional organization of HD cell tuning in thalamo-parahippocampal circuitry.

Quantum chemical investigation of electronic transitions of mitorubrin azaphilones.

Fungal azaphilones are a broad class of naturally-occurring pigments with diverse applications. Among the azaphilone pigments, mitorubrins are well recognized for their antiviral, antibacterial, antifungal, antiprotozoal, antidiabetic, and antiaging activities in addition to their well-known yellow-orange color. This makes these pigments interesting candidates for use in foods, as cosmetics, and as medicines. In particular, if it is desired to modify the properties of mitorubrin-based pigments, for example by derivatization, it is essential to have an understanding of the electronic spectra of the parent molecules. We have therefore undertaken a computational study of a series of mitorubrins, comparing our computed results with experimental UV/visible spectra. Both density-functional theory (DFT) and coupled-cluster (CC2) methods have been used, and in general, the results are in very good agreement with observation. In order to provide a simple and useful picture of the spectra we analyze the stronger transitions in terms of natural transition orbitals (NTOs).

Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Precursor-Directed Biosynthesis of Antialgal Fluorinated Bacillamide Derivatives in Bacillus atrophaeus.

The bacillamides are a class of indole alkaloids produced by the Bacillus genus that possess significant antialgal activity. Incorporation of fluorine into the bacillamides was carried out using a precursor-directed biosynthesis approach, with 4-, 5-, and 6-fluorotryptophan added to growing cultures of Bacillus atrophaeus IMG-11. This yielded the corresponding fluorinated analogues of bacillamides A and C, in addition to new derivatives of the related metabolite N-acetyltryptamine, thus demonstrating a degree of plasticity in the bacillamide biosynthetic pathway. The bacillamide derivatives were tested for activity against bloom-forming algae, which revealed that fluorination could improve the antialgal activity of these compounds in a site-specific manner, with fluorination at the 6-position consistently resulting in improved activity.

A thalamo-parietal cortex circuit is critical for place-action coordination.

The anterior and lateral thalamus (ALT) contains head direction cells that signal the directional orientation of an individual within the environment. ALT has direct and indirect connections with the parietal cortex (PC), an area hypothesized to play a role in coordinating viewer-dependent and viewer-independent spatial reference frames. This coordination between reference frames would allow an individual to translate movements toward a desired location from memory. Thus, ALT-PC functional connectivity would be critical for moving toward remembered allocentric locations. This hypothesis was tested in rats with a place-action task that requires associating an appropriate action (left or right turn) with a spatial location. There are four arms, each offset by 90°, positioned around a central starting point. A trial begins in the central starting point. After exiting a pseudorandomly selected arm, the rat had to displace the correct object covering one of two (left versus right) feeding stations to receive a reward. For a pair of arms facing opposite directions, the reward was located on the left, and for the other pair, the reward was located on the right. Thus, each reward location had a different combination of allocentric location and egocentric action. Removal of an object was scored as correct or incorrect. Trials in which the rat did not displace any objects were scored as "no selection" trials. After an object was removed, the rat returned to the center starting position and the maze was reset for the next trial. To investigate the role of the ALT-PC network, muscimol inactivation infusions targeted bilateral PC, bilateral ALT, or the ALT-PC network. Muscimol sessions were counterbalanced and compared to saline sessions within the same animal. All inactivations resulted in decreased accuracy, but only bilateral PC inactivations resulted in increased non selecting, increased errors, and longer latency responses on the remaining trials. Thus, the ALT-PC circuit is critical for linking an action with a spatial location for successful navigation.

Expected and Unexpected Products from the Biochemical Oxidation of Bacterial Alkylquinolones with CYP4F11.

2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe. This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.

Deciphering the biotransformation mechanism of dialkylresorcinols by CYP4F11.

Cytochrome P450 enzymes (CYPs) are one of the most important classes of oxidative enzymes in the human body, carrying out metabolism of various exogenous and endogenous substrates. In order to expand the knowledge of these enzymes' specificity and to obtain new natural product derivatives, CYP4F11, a cytochrome P450 monooxygenase, was used in the biotransformation of dialkylresorcinols 1 and 2, a pair of antibiotic microbial natural products. This investigation resulted in four biotransformation products including two oxidative products: a hydroxylated derivative (3) and a carboxylic acid derivative (4). In addition, acetylated (5) and esterified products (6) were isolated, formed by further metabolism by endogenous yeast enzymes. Oxidative transformations were highly regioselective, and took place exclusively at the ω-position of the C-5 alkyl chain. Homology modeling studies revealed that optimal hydrogen bonding between 2 and the enzyme can only be established with the C-5 alkyl chain pointing towards the heme. The closely-related CYP4F12 was not capable of oxidizing the dialkylresorcinol 2. Modeling experiments rationalize these differences by the different shapes of the binding pockets with respect to the non-oxidized alkyl chain. Antimicrobial testing indicated that the presence of polar groups on the side-chains reduces the antibiotic activity of the dialkylresorcinols.

A Dual Catalyst Strategy for Controlling Aluminum Nanocrystal Growth.

The synthesis of Al nanocrystals (Al NCs) is a rapidly expanding field, but there are few strategies for size and morphology control. Here we introduce a dual catalyst approach for the synthesis of Al NCs to control both NC size and shape. By using one catalyst that nucleates growth more rapidly than a second catalyst whose ligands affect NC morphology during growth, one can obtain both size and shape control of the resulting Al NCs. The combination of the two catalysts (1) titanium isopropoxide (TIP), for rapid nucleation, and (2) Tebbe's reagent, for specific facet-promoting growth, yields {100}-faceted Al NCs with tunable diameters between 35 and 65 nm. This dual-catalyst strategy could dramatically expand the possible outcomes for Al NC growth, opening the door to new controlled morphologies and a deeper understanding of earth-abundant plasmonic nanocrystal synthesis.

Plasmon-Generated Solvated Electrons for Chemical Transformations.

Methods for generating solvated electrons─free electrons in solution─have focused primarily on alkali metal ionization or high-energy electrons or photons. Here we report the generation of solvated electrons by exciting the plasmon resonance of Al nanocrystals suspended in solution with visible light. Two chemical reactions were performed: a radical-addition reaction with the spin-trap 2-methyl-2-nitrosopropane, and a model cyclization reaction with the radical clock 6-bromohex-1-ene. A quantum efficiency of at least ∼1.1% for plasmon absorbed photon to solvated electron generation can be inferred from the measured radical clock reaction concentration. This study demonstrates a simple way to generate solvated electrons for driving reductive organic chemical reactions in a quantifiable and controlled manner.

Sexually dimorphic organization of open field behavior following moderate prenatal alcohol exposure.

BACKGROUND: Prenatal alcohol exposure (PAE) can produce deficits in a wide range of cognitive functions but is especially detrimental to behaviors requiring accurate spatial information processing. In open field environments, spatial behavior is organized such that animals establish "home bases" marked by long stops focused around one location. Progressions away from the home base are circuitous and slow, while progressions directed toward the home base are non-circuitous and fast. The impact of PAE on the organization of open field behavior has not been experimentally investigated. METHODS: In the present study, adult female and male rats with moderate PAE or saccharin exposure locomoted a circular high walled open field for 30 minutes under lighted conditions. RESULTS: The findings indicate that PAE and sex influence the organization of open field behavior. Consistent with previous literature, PAE rats exhibited greater locomotion in the open field. Novel findings from the current study indicate that PAE and sex also impact open field measures specific to spatial orientation. While all rats established a home base on the periphery of the open field, PAE rats, particularly males, exhibited significantly less clustered home base stopping with smaller changes in heading between stops. PAE also impaired progression measures specific to distance estimation, while sex alone impacted progression measures specific to direction estimation. CONCLUSIONS: These findings support the conclusion that adult male rats have an increased susceptibility to the effects of PAE on the organization of open field behavior.

Soft particles facilitate flow of rigid particles in a 2D hopper.

The flow of granular materials through narrow openings governs flow and process efficiency in a variety of industrial settings. As the use of soft particles and other soft micro-materials becomes more widespread in consumer products, we seek to understand characteristics of granular flows beyond powder flows. We study clogging through a 2D hopper in systems consisting of a combination of soft and rigid particles of different sizes and mixing fractions. Our experimental results show that soft particles play a lubricating role in the flow of rigid spheres due to their deformability and slick surface, but the size of rigid particles influences clogging more than the size of soft ones. We simulate our results using a modification of the Durian bubble model to accommodate mixtures of particles of different softness. Without any adjustable parameters, the simulation results capture the clogging probability of soft-rigid particle mixtures through a 2D hopper.

Experimental and Computational Studies on the Biotransformation of Pseudopyronines with Human Cytochrome P450 CYP4F2.

The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV-vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1-3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

Case Report: Dry Eye Management Leads to Early Diagnosis of Seventh Nerve Schwannoma.

SIGNIFICANCE: Facial nerve schwannomas are rare tumors that are usually benign and relatively slow in their progression. Common symptoms include facial neuropathy, auditory deficiencies, and parotid masses. Because of slow progression, symptoms are often present for over a year before an appropriate diagnosis is made. PURPOSE: The purpose of this study was to present a case in which comprehensive dry eye assessments and management led to diagnosis of facial nerve shwannoma in a patient who had no presenting symptoms of auditory deficiencies or facial weakness. CASE REPORT: A 36-year-old woman presented for a contact lens examination with concerns of progressively worsening symptoms of irritation and dryness in her right eye that began 6 months earlier. Dry eye assessment visits and management strategies were implemented. Although this regimen was initially successful, symptoms returned after 7 months. At this visit, a new finding of incomplete blink in her right eye was manifested. Further in-office assessments revealed a weakened right orbicularis oculi and right frontalis muscle. These findings, combined with patient risk factors and no history of trauma, prompted MRI of the seventh nerve. Imaging revealed the presence of seventh nerve schwannoma, the location of which correlated with the geniculate ganglion and greater superficial petrosal nerve. This patient was later referred to a neurosurgeon, who assessed the risks and benefits of schwannoma removal. It was decided that the risks of the surgery outweighed the benefits, and regular MRI was scheduled for monitoring purposes. Appropriate management of the patient's chronic dry eye disease continues. CONCLUSIONS: Facial nerve schwannomas can and do present with no presenting subjective facial neuropathy symptoms aside from mild unilateral dry eye and can be detected with comprehensive dry eye management. This case may contribute to future minor adjustments in clinical practice guidelines for asymmetric dry eye assessments.

Aluminum Nanocrystals Grow into Distinct Branched Aluminum Nanowire Morphologies.

Plasmonic nanowires (NWs) have generated great interest in their applications in nanophotonics and nanotechnology. Here we report the synthesis of Al nanocrystals (NCs) with controlled morphologies that range from nanospheres to branched NW and NW bundles. This is accomplished by catalyzing the pyrolysis of triisobutyl aluminum (TIBA) with Tebbe's reagent, a titanium(III) catalyst with two cyclopentadienyl ligands. The ratio of TIBA to Tebbe's reagent is critical in determining the morphology of the resulting Al NC. The branched Al NWs grow in their ⟨100⟩ directions and are formed by oriented attachment of isotropic Al NCs on their {100} facets. Branched NWs are strongly absorptive from the UV to the mid-IR, with longitudinal dipolar, higher-order, and transverse plasmons, all contributing to their broadband response. This rapid Al NW synthesis enables the expanded use of Al for plasmonic and nanophotonic applications in the ultraviolet, visible, and infrared regions of the spectrum.

Site-Selective Nanoreactor Deposition on Photocatalytic Al Nanocubes.

Photoactivation of catalytic materials through plasmon-coupled energy transfer has created new possibilities for expanding the scope of light-driven heterogeneous catalysis. Here we present a nanoengineered plasmonic photocatalyst consisting of catalytic Pd islands preferentially grown on vertices of Al nanocubes. The regioselective Pd deposition on Al nanocubes does not rely on complex surface ligands, in contrast to site-specific transition-metal deposition on gold nanoparticles. We show that the strong local field enhancement on the sharp nanocube vertices provides a mechanism for efficient coupling of the plasmonic Al antenna to adjacent Pd nanoparticles. A substantial increase in photocatalytic H2 dissociation on Pd-bound Al nanocubes relative to pristine Al nanocubes can be observed, incentivizing further engineering of heterometallic antenna-reactor photocatalysts. Controlled growth of catalytic materials on plasmonic hot spots can result in more efficient use of the localized surface plasmon energy for photocatalysis, while minimizing the amount and cost of precious transition-metal catalysts.

Acoustic Vibrations of Al Nanocrystals: Size, Shape, and Crystallinity Revealed by Single-Particle Transient Extinction Spectroscopy.

Acoustic vibrations in plasmonic nanoparticles, monitored by an all-optical means, have attracted significant increasing interest because they provide unique insight into the mechanical properties of these metallic nanostructures. Al nanostructures are a recently emerging alternative to noble metal nanoparticles, because their broad wavelength tunability and high natural abundance make them ideal for many potential applications. Here, we investigate the acoustic vibrations of individual Al nanocrystals using a combination of electron microscopy and single-particle transient extinction spectroscopy, made possible with a low-pulse energy, high sensitivity, and probe-wavelength-tunable, single-particle transient extinction microscope. For chemically synthesized, faceted Al nanocrystals, the observed vibration frequency scales with the inverse particle diameter. In contrast, triangularly shaped Al nanocrystals support two distinct frequencies, corresponding to their in- and out-of-plane breathing modes. Unlike ensemble measurements, which measure average properties, measuring the damping time of the acoustic vibrations for individual particles enables us to investigate variations of the quality factor on the particle-to-particle level. Surprisingly, we find a large variation in quality factors even for nanocrystals of similar size and shape. This observed heterogeneity appears to result from substantially varying degrees of nanoparticle crystallinity even for chemically synthesized nanocrystals.

Synthesis of Natural and Unnatural Quinolones Inhibiting the Growth and Motility of Bacteria.

Synthesis of a recently discovered S-methylated quinolone natural product (1) was carried out, in addition to the production of a range of 2-substituted 4-quinolone derivatives (2-11). Two approaches were used: (i) the base-catalyzed cyclization of N-(ketoaryl)amides; (ii) attachment of the substituent to the quinolone core via a Suzuki-Miyaura cross-coupling. Also produced were a small suite of related 2(1H)-quinolones (12-19). The synthesized compounds were assessed for their antimicrobial properties. The alkene-substituted 4-quinolone 8 significantly inhibited the growth of a Pseudomonas aeruginosa strain, and both 4-quinolones and 2(1H)-quinolones were capable of inhibiting the swarming behavior of Bacillus subtilis.

Isolation, Identification, and Decomposition of Antibacterial Dialkylresorcinols from a Chinese Pseudomonas aurantiaca Strain.

A chemical investigation of a Chinese Pseudomonas aurantiaca strain has yielded a new benzoquinone (4) and furanone (5), in addition to the known dialkylresorcinols 1 and 2. Extensive decomposition studies on the major metabolite 1 produced an additional furanone derivative (6), a hydroxyquinone (7), and two unusual resorcinol and hydroxyquinone dimers (8 and 9). Structures were elucidated by nuclear magnetic resonance spectroscopy in combination with tandem mass spectrometry analysis. These studies illustrate the potential of artifacts as a source of additional chemical diversity. Compounds 1 and 2 showed moderate antibacterial activity against a panel of Gram-positive pathogens, while the antibacterial activities of the artifacts (4-9) were reduced.

Isolation of 2-Alkyl-4-quinolones with Unusual Side Chains from a Chinese Pseudomonas aeruginosa Isolate.

Chemical investigation of a Pseudomonas aeruginosa strain isolated from Hebei, China, led to the isolation of a suite of quinolones, quinolone-N-oxides, and phenazines, the structures of which were elucidated by detailed spectroscopic analysis. Most notable among the secondary metabolites isolated was an unprecedented 4-quinolone containing an S-methyl group in the side chain and a new derivative including a phenyl ring in the side chain, which expand significantly the variety of structural motifs found in the quinolones and raise interesting questions about their biosynthesis.