Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis.

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon.

Phase transition of GvpU regulates gas vesicle clustering in bacteria.

Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy. GV engineering has multiple applications, including reporter gene imaging, acoustic control and payload delivery. GVs often cluster into a honeycomb pattern to minimize occupancy of the cytosol.

50-nm Gas-Filled Protein Nanostructures to Enable the Access of Lymphatic Cells by Ultrasound Technologies.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need for microbubbles, which cannot transverse many biological barriers due to their large size. Here, the authors introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles(GVs) that are referred to as GVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to the authors' knowledge, the smallest stable, free-floating bubbles made to date.

Client satisfaction with antenatal care among clinic attendees in a tertiary health institution in Calabar, Cross River State, Nigeria.

Antenatal care is an important entry point to the health system and provides access to essential obstetric care. Satisfaction with different aspects of antenatal care, has the potential to improve health. This study aimed to determine the prevalence of satisfaction with Antenatal care services and identify factors influencing client satisfaction among antenatal clinic attendees in University of Calabar Teaching Hospital Calabar (UCTH), Cross River State.

50-nm gas-filled protein nanostructures to enable the access of lymphatic cells by ultrasound technologies.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as GVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to our knowledge, the smallest stable, free-floating bubbles made to date.

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET.

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown.

Measuring gas vesicle dimensions by electron microscopy.

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications.

Genetically Encodable Contrast Agents for Optical Coherence Tomography.

Optical coherence tomography (OCT) has gained wide adoption in biological research and medical imaging due to its exceptional tissue penetration, 3D imaging speed, and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT.

Recombinantly Expressed Gas Vesicles as Nanoscale Contrast Agents for Ultrasound and Hyperpolarized MRI.

Ultrasound and hyperpolarized magnetic resonance imaging enable the visualization of biological processes in deep tissues. However, few molecular contrast agents are available to connect these modalities to specific aspects of biological function. We recently discovered that a unique class of gas-filled protein nanostructures known as gas vesicles could serve as nanoscale molecular reporters for these modalities.

Protein Nanostructures Produce Self-Adjusting Hyperpolarized Magnetic Resonance Imaging Contrast through Physical Gas Partitioning.

Signal amplification strategies are critical for overcoming the intrinsically poor sensitivity of nuclear magnetic resonance (NMR) reporters in noninvasive molecular detection. A mechanism widely used for signal enhancement is chemical exchange saturation transfer (CEST) of nuclei between a dilute sensing pool and an abundant detection pool. However, the dependence of CEST amplification on the relative size of these spin pools confounds quantitative molecular detection with a larger detection pool typically making saturation transfer less efficient.

Biomolecular Ultrasound and Sonogenetics.

Visualizing and modulating molecular and cellular processes occurring deep within living organisms is fundamental to our study of basic biology and disease. Currently, the most sophisticated tools available to dynamically monitor and control cellular events rely on light-responsive proteins, which are difficult to use outside of optically transparent model systems, cultured cells, or surgically accessed regions owing to strong scattering of light by biological tissue. In contrast, ultrasound is a widely used medical imaging and therapeutic modality that enables the observation and perturbation of internal anatomy and physiology but has historically had limited ability to monitor and control specific cellular processes.

Proteins, air and water: reporter genes for ultrasound and magnetic resonance imaging.

A long-standing goal of molecular imaging is to visualize cellular function within the context of living animals, necessitating the development of reporter genes compatible with deeply penetrant imaging modalities such as ultrasound and magnetic resonance imaging (MRI). Until recently, no reporter genes for ultrasound were available, and most genetically encoded reporters for MRI were limited by metal availability or relatively low sensitivity. Here we review how these limitations are being addressed by recently introduced reporter genes based on air-filled and water-transporting biomolecules.

Acoustically modulated magnetic resonance imaging of gas-filled protein nanostructures.

Non-invasive biological imaging requires materials capable of interacting with deeply penetrant forms of energy such as magnetic fields and sound waves. Here, we show that gas vesicles (GVs), a unique class of gas-filled protein nanostructures with differential magnetic susceptibility relative to water, can produce robust contrast in magnetic resonance imaging (MRI) at sub-nanomolar concentrations, and that this contrast can be inactivated with ultrasound in situ to enable background-free imaging. We demonstrate this capability in vitro, in cells expressing these nanostructures as genetically encoded reporters, and in three model in vivo scenarios.

Biomolecular MRI reporters: Evolution of new mechanisms.

Magnetic resonance imaging (MRI) is a powerful technique for observing the function of specific cells and molecules inside living organisms. However, compared to optical microscopy, in which fluorescent protein reporters are available to visualize hundreds of cellular functions ranging from gene expression and chemical signaling to biomechanics, to date relatively few such reporters are available for MRI. Efforts to develop MRI-detectable biomolecules have mainly focused on proteins transporting paramagnetic metals for T and T relaxation enhancement or containing large numbers of exchangeable protons for chemical exchange saturation transfer.

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI.

Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques.

Characterizing Single Polymeric and Protein Nanoparticles with Surface Plasmon Resonance Imaging Measurements.

Near-infrared surface plasmon resonance imaging (SPRI) microscopy is used to detect and characterize the adsorption of single polymeric and protein nanoparticles (PPNPs) onto chemically modified gold thin films in real time. The single-nanoparticle SPRI responses, Δ%R, from several hundred adsorbed nanoparticles are collected in a single SPRI adsorption measurement. Analysis of Δ%R frequency distribution histograms is used to provide information on the size, material content, and interparticle interactions of the PPNPs.

A multicenter prospective study of neonatal outcomes at less than 32 weeks associated with indications for maternal admission and delivery.

Background: Counseling for patients with impending premature delivery traditionally has been based primarily on the projected gestational age at delivery. There are limited data regarding how the indications for the preterm birth affect the neonatal outcome and whether this issue should be taken into account in decisions regarding management and patient counseling.

Objective: We performed a prospective study of pregnancies resulting in premature delivery at less than 32 weeks to determine the influence of both the indications for admission and their associated indications for delivery on neonatal mortality and complications of prematurity.

NMR Hyperpolarization Techniques of Gases.

Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection.

17-hydroxyprogesterone caproate for preterm rupture of the membranes: a multicenter, randomized, double-blind, placebo-controlled trial.

Objective: Preterm rupture of membranes (PROM) is associated with an increased risk of preterm birth and neonatal morbidity. Prophylactic 17-hydroxyprogesterone caproate (17OHP-C) reduces the risk of preterm birth in some women who are at risk for preterm birth. We sought to test whether 17OHP-C would prolong pregnancy or improve perinatal outcome when given to mothers with preterm rupture of the membranes.

Structure of the membrane protein MerF, a bacterial mercury transporter, improved by the inclusion of chemical shift anisotropy constraints.

MerF is a mercury transport membrane protein from the bacterial mercury detoxification system. By performing a solid-state INEPT experiment and measuring chemical shift anisotropy frequencies in aligned samples, we are able to improve on the accuracy and precision of the initial structure that we presented. MerF has four N-terminal and eleven C-terminal residues that are mobile and unstructured in phospholipid bilayers.

NMR structures of membrane proteins in phospholipid bilayers.

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions.

Removal versus retention of cerclage in preterm premature rupture of membranes: a randomized controlled trial.

Objective: The decision of whether to retain or remove a previously placed cervical cerclage in women who subsequently rupture fetal membranes in a premature gestation is controversial and all studies to date are retrospective. We performed a multicenter randomized controlled trial of removal vs retention of cerclage in these patients to determine whether leaving the cerclage in place prolonged gestation and/or increased the risk of maternal or fetal infection.

Study Design: A prospective randomized multicenter trial of 27 hospitals was performed.

Mechanism of dilute-spin-exchange in solid-state NMR.

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter.

Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR.

Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances.