Neuron specific quantitation of Gαolf expression and signaling in murine brain tissue.

The heterotrimeric G-protein α subunit, Gαolf, acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gαolf, have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gαolf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gαolf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gαolf signaling in vivo in neurons that highly express Gnal.

G α olf Regulates Biochemical Signaling in Neurons Associated with Movement Control and Initiation.

The heterotrimeric G-protein α subunit, Gα olf , acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gα olf , have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gα olf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS . Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gα olf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL , with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gα olf signaling in vivo in neurons that highly express Gnal .

Chemical and Biological Characterization of Green and Processed Coffee Beans from Coffea arabica Varieties.

Coffee is one of the most consumed beverages in the world; its production is based mainly on varieties of the Coffea arabica species. Mexico stands out for its specialty and organic coffee. In Guerrero, the production is done by small indigenous community cooperatives that market their product as raw material. Official Mexico Standards stipulate the requirements for its commercialization within the national territory. In this work, the physical, chemical, and biological characterizations of green, medium, and dark roasted beans from C. arabica varieties were carried out. Analysis by HPLC showed higher chlorogenic acid (55 mg/g) and caffeine (1.8 mg/g) contents in the green beans of the Bourbon and Oro Azteca varieties. The caffeine (3.88 mg/g) and melanoidin (97 and 29 mg/g) contents increased according to the level of roasting; a dissimilar effect was found in the chlorogenic acid content (14.5 mg/g). The adequate nutritional content and the sensory evaluation allowed the classification of dark-roasted coffee as premium coffee (84.25 points) and medium-roasted coffee as specialty coffee (86.25 points). The roasted coffees presented antioxidant activity without cytotoxic effects; the presence of CGA and caffeine supports the beneficial effects of drinking coffee. The results obtained will serve as a basis for making decisions on improvements to the coffees analyzed.

SARS-CoV-2 Main Protease Targets Host Selenoproteins and Glutathione Biosynthesis for Knockdown via Proteolysis, Potentially Disrupting the Thioredoxin and Glutaredoxin Redox Cycles.

Associations between dietary selenium status and the clinical outcome of many viral infections, including SARS-CoV-2, are well established. Multiple independent studies have documented a significant inverse correlation between selenium status and the incidence and mortality of COVID-19. At the molecular level, SARS-CoV-2 infection has been shown to decrease the expression of certain selenoproteins, both in vitro and in COVID-19 patients. Using computational methods, our group previously identified a set of six host proteins that contain potential SARS-CoV-2 main protease (Mpro) cleavage sites. Here we show experimentally that Mpro can cleave four of the six predicted target sites, including those from three selenoproteins: thioredoxin reductase 1 (TXNRD1), selenoprotein F, and selenoprotein P, as well as the rate-limiting enzyme in glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC). Cleavage was assessed by incubating recombinant SARS-CoV-2 Mpro with synthetic peptides spanning the proposed cleavage sites, and analyzing the products via UPLC-MS. Furthermore, upon incubation of a recombinant Sec498Ser mutant of the full TXNRD1 protein with SARS-CoV-2 Mpro, the predicted cleavage was observed, destroying the TXNRD1 C-terminal redox center. Mechanistically, proteolytic knockdown of both TXNRD1 and GCLC is consistent with a viral strategy to inhibit DNA synthesis, conserving the pool of ribonucleotides for increased virion production. Viral infectivity could also be enhanced by GCLC knockdown, given the ability of glutathione to disrupt the structure of the viral spike protein via disulfide bond reduction. These findings shed new light on the importance of dietary factors like selenium and glutathione in COVID-19 prevention and treatment.

In Silico-Based Design and In Vivo Evaluation of an Anthranilic Acid Derivative as a Multitarget Drug in a Diet-Induced Metabolic Syndrome Model.

Metabolic syndrome (MetS) is a complex disease that affects almost a quarter of the world's adult population. In MetS, diabetes, obesity, hyperglycemia, high cholesterol, and high blood pressure are the most common disorders. Polypharmacy is the most used strategy for managing conditions related to MetS, but it has drawbacks such as low medication adherence. Multitarget ligands have been proposed as an interesting approach to developing drugs to treat complex diseases. However, suitable preclinical models that allow their evaluation in a context closer to a clinical situation of a complex disease are needed. From molecular docking studies, compound 1b, a 5-aminoanthranilic acid derivative substituted with 4'-trifluoromethylbenzylamino and 3',4'-dimethoxybenzamide moieties, was identified as a potential multitarget drug, as it showed high in silico affinity against targets related to MetS, including PPAR-α, PPAR-γ, and HMG-CoA reductase. It was evaluated in a diet-induced MetS rat model and simultaneously lowered blood pressure, glucose, total cholesterol, and triglyceride levels after a 14-day treatment. No toxicity events were observed during an acute lethal dose evaluation test at 1500 mg/kg. Hence, the diet-induced MetS model is suitable for evaluating treatments for MetS, and compound 1b is an attractive starting point for developing multitarget drugs.

Distinct roles of XPF-ERCC1 and Rad1-Rad10-Saw1 in replication-coupled and uncoupled inter-strand crosslink repair.

Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair.

DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination.

Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins.

Exonuclease 1 (Exo1) is a 5'→3' exonuclease and 5'-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.

High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging.

Single-molecule studies of protein-DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains-a method for organizing arrays of DNA molecules on a fluid lipid bilayer-has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900,000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3-a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.

Apyrase suppression raises extracellular ATP levels and induces gene expression and cell wall changes characteristic of stress responses.

Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.

Detecting secondary structure and surface orientation of helical peptide monolayers from resonant hybridization signals.

Hybridization of dominant vibrational modes with meta-surface resonance allows detection of both structural changes and surface orientations of bound helical peptides. Depending on the resonance frequency of meta-molecules, a red- or blue- shift in peptide Amide-I frequency is observed. The underlying coupling mechanism is described by using a temporal coupled mode theory that is in very good agreement with the experimental results. This hybridization phenomenon constitutes the basis of many nanophotonic systems such as tunable coupled mode bio-sensors and dynamic peptide systems driven by infrared signals.

Demonstration of α-helical structure of peptides tethered to gold surfaces using surface infrared and circular dichroic spectroscopies.

Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.

Tethering hydrophobic peptides to functionalized self-assembled monolayers on gold through two chemical linkers using the Huisgen cycloaddition.

Gold surfaces functionalized with an α-helical peptide have been generated by reacting an azide-terminated self-assembled monolayer with structured peptides containing two cyanophenylalanines through a Huisgen cycloaddition. Mixed monolayers of a reactive bromine-terminated thiol and inert alkane thiol were prepared at various concentrations of the Br-terminated moiety. These were reacted with sodium azide to form azide-terminated monolayers with controlled concentration of the reactive azide. These surfaces were studied through ellipsometry and X-ray photoelectron spectroscopy, which demonstrated that the concentration of the reactive azide group on the surface is controlled by the chemical conditions under which the monolayer is prepared. Grazing incident angle surface infrared spectroscopy (GRAS-IR) of the azide-terminated surface demonstrated that the azide is approximately perpendicular to the plane of the surface, as expected. These surfaces were then exposed to an α-helical peptide composed of alternating leucine and lysine residues, with two residues replaced with cyanophenylalanine to react with two neighboring surface-bound azide groups to bind the peptide to the surface through two covalent bonds. The yield of this reaction was quantified through monitoring the absorption of the azide group by GRAS-IR. Despite damage to the monolayer during the reaction, reaction yields of 80-98% were determined for optimized reaction conditions. Although the peptide retains its α-helical configuration under the reaction conditions, GRAS-IR analysis of the amide I and II modes of the surface-bound peptide showed that it is probably randomly oriented on the surface.

Simple access to the coronary venous system for left ventricular pacing.

Implantation of the LV lead for biventricular pacing can be challenging, time consuming, and often requires extensive fluoroscopy time. A conventional diagnostic 5 Fr left Amplatz catheter was used to cannulate the coronary sinus in 15 consecutive patients undergoing implantation of a biventricular pacemaker. When the coronary sinus was cannulated, the proximal end of the Amplatz catheter was cut and the coronary sinus sheath was passed over the Amplatz catheter that was then removed. Coronary sinus cannulation was achieved in all 15 patients with a mean fluoroscopy time of 3.34 +/- 1.9 minutes. Subsequent implantation of a biventricular pacemaker was successful and free of complications in all the 15 patients.