Coherent reflection recovery in scattering from the ocean surface using the frequency-difference autoproducta).

The coherence of rough sea-surface-scattered acoustic fields decreases with increasing frequency. The frequency-difference autoproduct, a quadratic product of acoustic fields at nearby frequencies, mimics a genuine field at the difference frequency. In rough-surface scattering, the autoproduct's lower effective frequency decreases the apparent surface roughness, restoring coherent reflection. Herein, the recovery of coherent reflection in sea surface scattering via the frequency-difference autoproduct is examined for data collected off the coast of New Jersey during the Shallow Water '06 (SW06) experiment. An acoustic source at depth 40 m and receiver at depth 24.3 m and range 200 m interrogated 160 independent realizations of the ocean surface. The root mean square surface height h was 0.167 m, and broadcast frequencies were 14-20 kHz, so that 2.5 ≤kh cos θ≤ 3.7 for acoustic wavenumber k and incidence angle θ. Measured autoproducts, constructed from scattered constituent fields, show significant coherent reflection at sufficiently low difference frequencies. Theoretical results, using the Kirchhoff approximation and a non-analytic surface autocorrelation function, agree with experimental findings. The match is improved using a numerical strategy, exploiting the relationship between autoproduct-based coherence recovery, the ocean-surface autocorrelation function, and the ocean-surface height spectrum. Error bars computed from Monte Carlo scattering simulations support the validity of the measured coherence recovery.

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Active intensity vortex and stagnation point singularities in a shallow underwater waveguide.

Vector acoustic properties of a narrowband acoustic field are observed as a function of range from a source towed in waters of depth 77 m on the New England Mud Patch. At the source frequency (43 Hz), the waveguide supported three trapped modes, with mode 2 weakly excited owing to the towed source depth. The receiving sensor was positioned 1.45 m above the seafloor with a sampling range aperture of 2500 m. The vector acoustics observations enabled study of vortex regions that encompass two singular points for active acoustic intensity: the vortex point, which is co-located with a dislocation, and stagnation point. Interpretative modeling, based on the normal modes and using a geoacoustic model consistent with those emerging from studies conducted at this location, is in agreement with these measurements. Model-data comparisons were based on the first-order variables of acoustic pressure and velocity along with inverse Hankel transforms, which yield normalized horizontal wavenumber spectra, and second-order variables in the form of horizontal and vertical intensity as well as non-dimensional intensity-based ratios. These measures provide a degree of observational confirmation of some vortex region properties. Both observations and modeling point to a gradual deepening of such regions with increasing range owing to sediment attenuation.

A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography.

Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via light-weight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Šresolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.

Potential and kinetic energy of underwater noise measured below a passing ship and response to sub-bottom layering.

Observed near the seafloor, broadband noise emissions from a vessel passing directly above exhibit frequency bands where potential acoustic energy is greater than kinetic energy while the opposite occurs in neighboring frequency bands. The condition where the dynamic and kinematic energy forms differ in this manner is characteristic to interference involving steep angles or near-normal incidence reflection from the seafloor. Measurements are made at two experimental sites using a research vessel passing above a vector sensor, positioned ∼1.5 m above the seabed, resulting in a vessel horizontal range approaching ∼0. The data are expressed as a ratio of kinetic to potential energy in decibels and yield information on seabed properties. A model for kinetic and potential energy is developed from the method of images using a layered seabed and is used to invert data collected in Puget Sound. A higher-impedance seabed is identified via inversion, which is consistent with the thin Holocene sediments in the region. For data collected on the New England Mud Patch, the model is instead applied directly to nominal seabed parameters originating from prior studies that identify a low-speed mud layer atop a higher-speed transition layer separating the mud substrate from a sediment basement.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction.

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

Physical effects of sound exposure from underwater explosions on Pacific mackerel (Scomber japonicus): Effects on the inner ear.

Studies of the effects of sounds from underwater explosions on fishes have not included examination of potential effects on the ear. Caged Pacific mackerel (Scomber japonicus) located at seven distances (between approximately 35 and 800 m) from a single detonation of 4.5 kg of C4 explosives were exposed. After fish were recovered from the cages, the sensory epithelia of the saccular region of the inner ears were prepared and then examined microscopically. The number of hair cell (HC) ciliary bundles was counted at ten preselected 2500 µm2 regions. HCs were significantly reduced in fish exposed to the explosion as compared to the controls. The extent of these differences varied by saccular region, with damage greater in the rostral and caudal ends and minimal in the central region. The extent of effect also varied in animals at different distances from the explosion, with damage occurring in fish as far away as 400 m. While extrapolation to other species and other conditions (e.g., depth, explosive size, and distance) must be performed with extreme caution, the effects of explosive sounds should be considered when environmental impacts are estimated for marine projects.

Physical effects of sound exposure from underwater explosions on Pacific mackerel (Scomber japonicus): Effects on non-auditory tissues.

Underwater explosions from activities such as construction, demolition, and military activities can damage non-auditory tissues in fishes. To better understand these effects, Pacific mackerel (Scomber japonicus) were placed in mid-depth cages with water depth of approximately 19.5 m and exposed at distances of 21 to 807 m to a single mid-depth detonation of C4 explosive (6.2 kg net explosive weight). Following exposure, potential correlations between blast acoustics and observed physical effects were examined. Primary effects were damage to the swim bladder and kidney that exceeded control levels at ≤333 m from the explosion [peak sound pressure level 226 dB re 1 µPa, sound exposure level (SEL) 196 dB re 1 µPa2 s, pressure impulse 98 Pa s]. A proportion of fish were dead upon retrieval at 26-40 min post exposure in 6 of 12 cages located ≤157 m from the explosion. All fish that died within this period suffered severe injuries, especially swim bladder and kidney rupture. Logistic regression models demonstrated that fish size or mass was not important in determining susceptibility to injury and that peak pressure and SEL were better predictors of injury than was pressure impulse.

Vector acoustic and polarization properties of underwater ship noise.

Vector acoustic field properties measured during the 2017 Seabed Characterization Experiment (SBCEX17) are presented. The measurements were made using the Intensity Vector Autonomous Recorder (IVAR) that records acoustic pressure and acceleration from which acoustic velocity is obtained. Potential and kinetic energies of underwater noise from two ship sources, computed in decidecimal bands centered between 25-630 Hz, are equal within calibration uncertainty of ±1.5 dB, representing a practical result towards the inference of kinematic properties from pressure-only measurements. Bivariate signals limited to two acoustic velocity components are placed in the context of the Stokes framework to describe polarization properties, such as the degree of polarization, which represents a statistical measure of the dispersion of the polarization properties. A bivariate signal composed of vertical and radial velocity components within a narrow frequency band centered at 63 Hz representing different measures of circularity and degree of polarization is examined in detail, which clearly demonstrates properties of bivariate signal trajectory. An examination of the bivariate signal composed of the two horizontal components of velocity within decidecimal bands centered at 63 Hz and 250 Hz demonstrates the importance of the degree of polarization in bearing estimation of moving sources.

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Šresolution when exposed to XFEL radiation, which is an improvement of 0.15 Šover previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

A 300-fold conductivity increase in microbial cytochrome nanowires due to temperature-induced restructuring of hydrogen bonding networks.

Although proteins are considered as nonconductors that transfer electrons only up to 1 to 2 nanometers via tunneling, Geobacter sulfurreducens transports respiratory electrons over micrometers, to insoluble acceptors or syntrophic partner cells, via nanowires composed of polymerized cytochrome OmcS. However, the mechanism enabling this long-range conduction is unclear. Here, we demonstrate that individual nanowires exhibit theoretically predicted hopping conductance, at rate (>1010 s-1) comparable to synthetic molecular wires, with negligible carrier loss over micrometers. Unexpectedly, nanowires show a 300-fold increase in their intrinsic conductance upon cooling, which vanishes upon deuteration. Computations show that cooling causes a massive rearrangement of hydrogen bonding networks in nanowires. Cooling makes hemes more planar, as revealed by Raman spectroscopy and simulations, and lowers their reduction potential. We find that the protein surrounding the hemes acts as a temperature-sensitive switch that controls charge transport by sensing environmental perturbations. Rational engineering of heme environments could enable systematic tuning of extracellular respiration.

Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.

Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis.

Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis.

Polarization of ocean acoustic normal modes.

In ocean acoustics, shallow water propagation is conveniently described using normal mode propagation. This article proposes a framework to describe the polarization of normal modes, as measured using a particle velocity sensor in the water column. To do so, the article introduces the Stokes parameters, a set of four real-valued quantities widely used to describe polarization properties in wave physics, notably for light. Stokes parameters of acoustic normal modes are theoretically derived, and a signal processing framework to estimate them is introduced. The concept of the polarization spectrogram, which enables the visualization of the Stokes parameters using data from a single vector sensor, is also introduced. The whole framework is illustrated on simulated data as well as on experimental data collected during the 2017 Seabed Characterization Experiment. By introducing the Stokes framework used in many other fields, the article opens the door to a large set of methods developed and used in other contexts but largely ignored in ocean acoustics.

Trends in low-frequency underwater noise off the Oregon coast and impacts of COVID-19 pandemic.

Approximately six years of underwater noise data recorded from the Regional Cabled Array network are examined to study long-term trends. The data originate from station HYS14 located 87 km offshore of Newport, OR. The results indicate that the third-octave band level centered at 63 Hz and attributable to shipping activity is reduced in the spring of 2020 by about 1.6 dB relative to the mean of the prior five years, owing to the reduced economic activity initiated by the COVID-19 pandemic. The results are subtle, as the noise reduction is less than the typical seasonal fluctuation associated with warming ocean surface temperatures in the summer that reduces mode excitation support at typical ship source depths, causing a repeated annual level change on the order of 4 dB at shipping frequencies. Seasonality of the noise contribution near 20 Hz from fin whales is also discussed. Corroboration of a COVID-19 effect on shipping noise is offered by an analysis of automatic identification system shipping data and shipping container activity for Puget Sound, over the same six-year period, which shows a reduction in the second quarter of 2020 by ∼19% and ∼17%, respectively, relative to the mean of the prior five years.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines.

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (105 s-1) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

Power Law Behavior in Protein Desorption Kinetics Originating from Sequential Binding and Unbinding.

The study of protein adsorption at the single molecule level has recently revealed that the adsorption is reversible, but with a long-tailed residence time distribution which can be approximated with a sum of exponential functions putatively related to distinct adsorption sites. Here it is proposed that the shape of the residence time distribution results from an adsorption process with sequential and reversible steps that contribute to overall binding strength resembling "zippering". In this model, the survival function of the residence time distribution of single proteins varies from an exponential distribution for a single adsorption step to a power law distribution with exponent -1/2 for a large number of adsorption steps. The adsorption of fluorescently labeled fibrinogen to glass surfaces is experimentally studied with single molecule imaging. The experimental residence time distribution can be readily fit by the proposed model. This demonstrates that the observed long residence times can arise from stepwise adsorption rather than rare but strong binding sites and provides guidance for the control of protein adsorption to biomaterials.