Numerical Investigation of Odor-Guided Navigation in Flying Insects: Impact of Turbulence, Wingbeat-Induced Flow, and Schmidt Number on Odor Plume Structures.

Odor-guided navigation is fundamental to the survival and reproductive success of many flying insects. Despite its biological importance, the mechanics of how insects sense and interpret odor plumes in the presence of complex flow fields remain poorly understood. This study employs numerical simulations to investigate the influence of turbulence, wingbeat-induced flow, and Schmidt number on the structure and perception of odor plumes by flying insects. Using an in-house computational fluid dynamics solver based on the immersed-boundary method, we solve the three-dimensional Navier-Stokes equations to model the flow field. The solver is coupled with the equations of motion for passive flapping wings to emulate wingbeat-induced flow. The odor landscape is then determined by solving the odor advection-diffusion equation. By employing a synthetic isotropic turbulence generator, we introduce turbulence into the flow field to examine its impact on odor plume structures. Our findings reveal that both turbulence and wingbeat-induced flow substantially affect odor plume characteristics. Turbulence introduces fluctuations and perturbations in the plume, while wingbeat-induced flow draws the odorant closer to the insect's antennae. Moreover, we demonstrate that the Schmidt number, which affects odorant diffusivity, plays a significant role in odor detectability. Specifically, at high Schmidt numbers, larger fluctuations in odor sensitivity are observed, which may be exploited by insects to differentiate between various odorant volatiles emanating from the same source. This study provides new insights into the complex interplay between fluid dynamics and sensory biology and behavior, enhancing our understanding of how flying insects successfully navigate using olfactory cues in turbulent environments.

OLD family nuclease function across diverse anti-phage defense systems.

Bacteriophages constitute a ubiquitous threat to bacteria, and bacteria have evolved numerous anti-phage defense systems to protect themselves. These systems include well-studied phenomena such as restriction endonucleases and CRISPR, while emerging studies have identified many new anti-phage defense systems whose mechanisms are unknown or poorly understood. Some of these systems involve overcoming lysogenization defect (OLD) nucleases, a family of proteins comprising an ABC ATPase domain linked to a Toprim nuclease domain. Despite being discovered over 50 years ago, OLD nuclease function remained mysterious until recent biochemical, structural, and bioinformatic studies revealed that OLD nucleases protect bacteria by functioning in diverse anti-phage defense systems including the Gabija system and retrons. In this review we will highlight recent discoveries in OLD protein function and their involvement in multiple discrete anti-phage defense systems.

Characterizing Aerosol Generating Procedures With Background Oriented Schlieren.

The potential for characterizing aerosol generating procedures (AGPs) using background oriented schlieren (BOS) flow visualization was investigated in two clinical situations. A human-scale BOS system was used on a manikin simulating jet ventilation and extubation. A novel approach to representation of the BOS images using line integral convolution allows direct evaluation of both magnitude and direction of the refractive index gradient field. Plumes issuing from the manikin's mouth were clearly visualized and characterized in both experiments, and it is recommended that BOS be adapted into a clinical tool for risk evaluation in clinical environments.

Borate Transporters and SLC4 Bicarbonate Transporters Share Key Functional Properties.

Borate transporters are membrane transport proteins that regulate intracellular borate levels. In plants, borate is a micronutrient essential for growth but is toxic in excess, while in yeast, borate is unnecessary for growth and borate export confers tolerance. Borate transporters share structural homology with human bicarbonate transporters in the SLC4 family despite low sequence identity and differences in transported solutes. Here, we characterize the S. cerevisiae borate transporter Bor1p and examine whether key biochemical features of SLC4 transporters extend to borate transporters. We show that borate transporters and SLC4 transporters share multiple properties, including lipid-promoted dimerization, sensitivity to stilbene disulfonate-derived inhibitors, and a requirement for an acidic residue at the solute binding site. We also identify several amino acids critical for Bor1p function and show that disease-causing mutations in human SLC4A1 will eliminate in vivo function when their homologous mutations are introduced in Bor1p. Our data help elucidate mechanistic features of Bor1p and reveal significant functional properties shared between borate transporters and SLC4 transporters.

Human SLC4A11 does not complement BOR1 or support borate transport in Saccharomyces cerevisiae.

Borate is an essential micronutrient in plants regulated by borate transporters, which also protect both yeast and plants from toxically high levels of borate and share homology with the human SLC4 transporters. SLC4A11 is linked to congenital hereditary endothelial dystrophy and was initially reported to transport borate before subsequent studies rebutted this conclusion. To better understand the transport activities of purported borate transporters, we tested the ability of SLC4A11 and eleven borate transporters from A. thaliana and O. sativa to complement a BOR1 deletion in S. cerevisiae . We show that AtBOR4 , AtBOR5 , AtBOR7 , OsBOR2 , and OsBOR3 can each complement ScBOR1 , while the rest of the transporters tested do not rescue growth. Additionally, quantification of intracellular borate content demonstrates that SLC4A11 does not export borate in yeast, supporting studies that its transported substrate is not borate.

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Depression-like symptoms of withdrawal in a genetic mouse model of binge methamphetamine intake.

Binge methamphetamine (MA) users have higher MA consumption, relapse rates and depression-like symptoms during early periods of withdrawal, compared with non-binge users. The impact of varying durations of MA abstinence on depression-like symptoms and on subsequent MA intake was examined in mice genetically prone to binge-level MA consumption. Binge-level MA intake was induced using a multiple-bottle choice procedure in which mice were offered one water drinking tube and three tubes containing increasing concentrations of MA in water, or four water tubes (control group). In two studies, depression-like symptoms were measured using a tail-suspension test and a subsequent forced-swim test, after forced abstinence of 6 and 30 hours from a 28-day course of chronic MA intake. An additional study measured the same depression-like symptoms, as well as MA intake, after prolonged abstinence of 1 and 2 weeks. MA high drinking mice and one of their progenitor strains DBA/2J escalated their MA intake with increasing MA concentration; however, MA high drinking mice consumed almost twice as much MA as DBA/2J mice. Depression-like symptoms were significantly higher early after MA access was withdrawn, compared to levels in drug-naïve controls, with more robust effects of MA withdrawal observed in MA high drinking than DBA/2J mice. When depression-like symptoms were examined after 1 or 2 weeks of forced abstinence in MA high drinking mice, depression-like symptoms dissipated, and subsequent MA intake was high. The MA high drinking genetic mouse model has strong face validity for human binge MA use and behavioral sequelae associated with abstinence.

Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers.

Boron is essential for plant growth because of its incorporation into plant cell walls; however, in excess it is toxic to plants. Boron transport and homeostasis in plants is regulated in part by the borate efflux transporter Bor1, a member of the solute carrier (SLC) 4 transporter family with homology to the human bicarbonate transporter Band 3. Here, we present the 4.1-Å resolution crystal structure of Arabidopsis thaliana Bor1. The structure displays a dimeric architecture in which dimerization is mediated by centralized Gate domains. Comparisons with a structure of Band 3 in an outward-open state reveal that the Core domains of Bor1 have rotated inwards to achieve an occluded state. Further structural comparisons with UapA, a xanthine transporter from the nucleobase-ascorbate transporter family, show that the downward pivoting of the Core domains relative to the Gate domains may access an inward-open state. These results suggest that the SLC4, SLC26, and nucleobase-ascorbate transporter families all share an elevator transport mechanism in which alternating access is provided by Core domains that carry substrates across a membrane.

Thermodynamic system drift in protein evolution.

Proteins from thermophiles are generally more thermostable than their mesophilic homologs, but little is known about the evolutionary process driving these differences. Here we attempt to understand how the diverse thermostabilities of bacterial ribonuclease H1 (RNH) proteins evolved. RNH proteins from Thermus thermophilus (ttRNH) and Escherichia coli (ecRNH) share similar structures but differ in melting temperature (T(m)) by 20 °C. ttRNH's greater stability is caused in part by the presence of residual structure in the unfolded state, which results in a low heat capacity of unfolding (ΔCp) relative to ecRNH. We first characterized RNH proteins from a variety of extant bacteria and found that Tm correlates with the species' growth temperatures, consistent with environmental selection for stability. We then used ancestral sequence reconstruction to statistically infer evolutionary intermediates along lineages leading to ecRNH and ttRNH from their common ancestor, which existed approximately 3 billion years ago. Finally, we synthesized and experimentally characterized these intermediates. The shared ancestor has a melting temperature between those of ttRNH and ecRNH; the T(m)s of intermediate ancestors along the ttRNH lineage increased gradually over time, while the ecRNH lineage exhibited an abrupt drop in Tm followed by relatively little change. To determine whether the underlying mechanisms for thermostability correlate with the changes in T(m), we measured the thermodynamic basis for stabilization--ΔCp and other thermodynamic parameters--for each of the ancestors. We observed that, while the T(m) changes smoothly, the mechanistic basis for stability fluctuates over evolutionary time. Thus, even while overall stability appears to be strongly driven by selection, the proteins explored a wide variety of mechanisms of stabilization, a phenomenon we call "thermodynamic system drift." This suggests that even on lineages with strong selection to increase stability, proteins have wide latitude to explore sequence space, generating biophysical diversity and potentially opening new evolutionary pathways.

Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.

The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage.

Type II topoisomerases are required for the management of DNA superhelicity and chromosome segregation, and serve as frontline targets for a variety of small-molecule therapeutics. To better understand how these enzymes act in both contexts, we determined the 2.9-Å-resolution structure of the DNA cleavage core of human topoisomerase IIα (TOP2A) bound to a doubly nicked, 30-bp duplex oligonucleotide. In accord with prior biochemical and structural studies, TOP2A significantly bends its DNA substrate using a bipartite, nucleolytic center formed at an N-terminal dimerization interface of the cleavage core. However, the protein also adopts a global conformation in which the second of its two inter-protomer contact points, one at the C-terminus, has separated. This finding, together with comparative structural analyses, reveals that the principal site of DNA engagement undergoes highly quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit-subunit interactions. Additional consideration of our TOP2A model in light of an etoposide-inhibited complex of human topoisomerase IIß (TOP2B) suggests possible modification points for developing paralog-specific inhibitors to overcome the tendency of topoisomerase II-targeting chemotherapeutics to generate secondary malignancies.

Design, Implementation, Simulation, and Visualization of a Highly Efficient RIM Microfluidic Mixer for Rapid Freeze-Quench of Biological Samples.

Rapid freeze-quench (RFQ) trapping of short-lived reaction intermediates for spectroscopic study plays an important role in the characterization of biological reactions. Recently there has been considerable effort to achieve submillisecond reaction deadtimes. We present here a new, robust, high-velocity microfluidic mixer that enables such rapid freeze-quenching. It is a based on the mixing method of two impinging jets commonly used in reaction injection molding (RIM) of plastics. This method achieves efficient mixing by inducing chaotic flow at relatively low Reynolds numbers (Re =140). We present the first mathematical simulation and microscopic visualization of mixing in such RFQ micromixers, the results of which show that the impinging solutions efficiently mix within the mixing chamber. These tests, along with a practical demonstration in a RFQ setup that involves copper wheels, show this new mixer can in practice provide reaction deadtimes as low as 100 microseconds.

All tangled up: how cells direct, manage and exploit topoisomerase function.

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.

A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.

Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clinical antibiotics and anti-cancer agents. These enzymes catalyse the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or 'gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyse G-segment cleavage. Recent efforts have proposed that strand scission relies on a 'two-metal mechanism', a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5A resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalysed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular 'trapdoor' to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.

Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function.

Prion diseases are fatal transmissible neurodegenerative diseases that result from structural conversion of the prion protein into a disease-associated isoform. The prion protein contains a single disulfide bond. Our analysis of all NMR structures of the prion protein (total of 440 structures over nine species) containing an explicit disulfide bond reveals that the bond exists predominantly in a stable low-energy state, but can also adopt a high-energy configuration. The side chains of two tyrosine residues and one phenylalanine residue control access of solvent to the disulfide bond. Notably, the side chains rotate away from the disulfide bond in the high-energy state, exposing the disulfide bond to solvent. The importance of these aromatic residues for protein function was analysed by mutating them to alanine residues and analysing the properties of the mutant proteins using biophysical and cell biological approaches. Whereas the mutant protein behaved similarly to wild-type prion protein in recombinant systems, the mutants were retained in the endoplasmic reticulum of mammalian cells and degraded by the proteasomal system. The cellular behaviour of the aromatic residue mutants was similar to the cellular behaviour of a disulfide bond mutant prion protein in which the cysteine residues were replaced with alanine, a result which is consistent with an unstable disulfide bond in the aromatic residue mutants. These observations suggest that the conformation of the prion protein disulfide bond may have implications for correct maturation and function of this protein.

A biochemical approach to identifying microRNA targets.

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.

Search for allosteric disulfide bonds in NMR structures.

BACKGROUND: Allosteric disulfide bonds regulate protein function when they break and/or form. They typically have a -RHStaple configuration, which is defined by the sign of the five chi angles that make up the disulfide bond. RESULTS: All disulfides in NMR and X-ray protein structures as well as in refined structure datasets were compared and contrasted for configuration and strain energy. CONCLUSION: The mean dihedral strain energy of 55,005 NMR structure disulfides was twice that of 42,690 X-ray structure disulfides. Moreover, the energies of all twenty types of disulfide bond was higher in NMR structures than X-ray structures, where there was an exponential decrease in the mean strain energy as the incidence of the disulfide type increased. Evaluation of protein structures for which there are X-ray and NMR models shows that the same disulfide bond can exist in different configurations in different models. A disulfide bond configuration that is rare in X-ray structures is the -LHStaple. In NMR structures, this disulfide is characterised by a particularly high potential energy and very short alpha-carbon distance. The HIV envelope glycoprotein gp120, for example, is regulated by thiol/disulfide exchange and contains allosteric -RHStaple bonds that can exist in the -LHStaple configuration. It is an open question which form of the disulfide is the functional configuration.

Allosteric disulfide bonds.

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.

Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC.

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.

An arginine to lysine mutation in the vicinity of the heme propionates affects the redox potentials of the hemes and associated electron and proton transfer in cytochrome c oxidase.

Cytochrome c oxidase pumps protons across a membrane using energy from electron transfer and reduction of oxygen to water. It is postulated that an element of the energy transduction mechanism is the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possible sites on which protons could reside, in addition to the conserved carboxylate E286, are the propionate groups of heme a and/or heme a(3). A highly conserved pair of arginines (R481 and R482) interact with these propionates through ionic and hydrogen bonds. This study shows that the conservative mutant, R481K, although as fully active as the wild type under many conditions, exhibits a significant decrease in the midpoint redox potential of heme a relative to Cu(A) (DeltaE(m)) of approximately equal 40 mV, has lowered activity under conditions of high pH or in the presence of a membrane potential, and has a slowed heme a(3) reduction with dithionite. Another mutant, D132A, which strongly inhibits proton uptake from the internal side of the membrane, has <4% of the activity of the wild type and appears to be dependent on proton uptake from the outside. A double mutation, D132A/R481K, is even more strongly inhibited ( approximately 1% of that of the wild type). The more-than-additive effect supports the concept that R481K not only lowers the midpoint potential of heme a but also limits a supply route for protons from the outside of the membrane used by the D132 mutant. The results are consistent with an important role of R481 and heme a/a(3) propionates in proton movement in a reversible exit path.