Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

Novel geometrically asymmetric biscinnamyl-sulfone compounds (6a-c) with donor-π-conjugated spacer-acceptor functionality were successfully synthesized. This was achieved by coupling cinnamaldehyde precursors with 3,3\'-diaminodiphenyl sulfone in dry organic solvents, resulting in high yields. Several spectroscopic techniques were employed to identify the derivatives. The absorption spectra of these compounds exhibited broad bands that spanned up to 120 nm, which can be attributed to their extended conjugation systems. In order to explore the electronic transitions of these materials, Time-Dependent Density-Functional Theory (TD-DFT) with EIFPCM solvation mode was utilized. We computationally investigated the static nonlinear optical (NLO) parameters, including dipole moments (μ), polarizability (α), anisotropic polarizability (Δα), first-order hyperpolarization (β), and second-order hyperpolarization (γ). Although the new structures possess different functional groups, they displayed similar electronic potentials when their molecular electrostatic potentials were plotted. These potentials are crucial in stabilizing the molecules in crystal systems through noncovalent forces such as C-H⋯π stacking and hydrogen bonding. They also provide insights into the electronic assessments and energetics of these individual forces. By estimating the frontier orbitals, we gained an understanding of the intramolecular charge transfer in the compounds. Energy gap values were determined using the orbitals of density of states method and experimentally via the Tauc method. The computational and experimental results were in good agreement. Lastly, we examined the influence of different protic and aprotic solvents on the absorption bands of compound 6b, as an example. This compound showed a significant bathochromic shift of 41 nm upon changing the solvent from acetic acid to dimethyl sulfoxide.

When mechanical stimulation was applied to free swimming Paramecium, forward swimming velocity transiently increased due to activation of the posterior mechanosensory channels. The behavior response, known as \"escape response,\" requires membrane hyperpolarization and the activation of K-channel type adenylate cyclases. Our hypothesis is that this escape response also involves activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. HCN channels are activated by hyperpolarization and are modulated by cyclic nucleotides such as cAMP and cGMP. They play a critical role in many excitable cells in higher animals. If HCN channels act in Paramecium, this should help to enhance and prolong hyperpolarization, thereby increasing the swimming speed of Paramecium. This study used RNAi to examine the role of the HCN channel 1 in the escape responses by generating hcn1-gene knockdown cells (hcn1-KD). These cells showed reduced mechanically-stimulated escape responses and a lack of cGMP-dependent increases in swimming speed. Electrophysiological experiments demonstrated reduced hyperpolarization upon injection of large negative currents in hcn1-KD cells. This is consistent with a decrease in HCN1 channel activity and changes in the escape response. These findings suggest that HCN1 channels are K+ channels that regulate the escape response of Paramecium by amplifying the hyperpolarizations elicited by posterior mechanical stimulation.

OBJECTIVE: To demonstrate the feasibility of 3D echo-planar spectroscopic imaging (EPSI) technique with rapid volumetric radial k-space sampling for hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) in vivo.
METHODS: A radial EPSI (rEPSI) was implemented on a 3 T clinical PET/MR system. To enable volumetric coverage, the sinusoidal shaped readout gradients per k-t-spoke were rotated along the three spatial dimensions in a golden-angle like manner. A distance-weighted, density-compensated gridding reconstruction was used, also in cases with undersampling of spokes in k-space. Measurements without and with HP 13C-labeled substances were performed in phantoms and rats using a double-resonant 13C/1H volume resonator with 72 mm inner diameter.
RESULTS: Phantom measurements demonstrated the feasibility of the implemented rEPSI sequence, as well as the robustness to undersampling in k-space up to a factor of 5 without advanced reconstruction techniques. Applied to measurements with HP [1-13C]pyruvate in a tumor-bearing rat, we obtained well-resolved MRSI datasets with a large matrix size of 123 voxels covering the whole imaging FOV of (180 mm)3 within 6.3 s, enabling to observe metabolism in dynamic acquisitions.
CONCLUSIONS: After further optimization, the proposed rEPSI method may be useful in applications of HP 13C-tracers where unknown or varying metabolite resonances are expected, and the acquisition of dynamic, volumetric MRSI datasets with an adequate temporal resolution is a challenge.

Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. DNPjuice comprising of glycerol-d8/D2O/H2O has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the invitro preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at ∼72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for 13C-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.

OBJECTIVE: The objective of the present work was to test the feasibility of in vivo imaging of hyperpolarized 50-nm silicon-29 (29Si) nanoparticles.
METHODS: Commercially available, crystalline 50-nm nanoparticles were hyperpolarized using dynamic polarization transfer via the endogenous silicon oxide-silicon defects without the addition of exogenous radicals. Phantom experiments were used to quantify the effect of sample dissolution and various surface coating on T1 and T2 relaxation. The in vivo feasibility of detecting hyperpolarized silicon-29 was tested following intraperitoneal, intragastric, or intratumoral injection in mice and compared with the results obtained with previously reported, large, micrometer-size particles. The tissue clearance of SiNPs was quantified in various organs using inductively coupled plasma optical emission spectroscopy.
RESULTS: In vivo images obtained after intragastric, intraperitoneal, and intratumoral injection compare favorably between small and large SiNPs. Improved distribution of small SiNPs was observed after intraperitoneal and intragastric injection as compared with micrometer-size SiNPs. Sufficient clearance of nanometer-size SiNPs using ex vivo tissue sample analysis was observed after 14 days following injection, indicating their safe use.
CONCLUSIONS: In vivo MRI of hyperpolarized small 50-nm SiNPs is feasible with polarization levels and room-temperature relaxation times comparable to large micrometer-size particles.

To understand the function of cells such as neurons within an organism, it can be instrumental to inhibit cellular function, or to remove the cell (type) from the organism, and thus to observe the consequences on organismic and/or circuit function and animal behavior. A range of approaches and tools were developed and used over the past few decades that act either constitutively or acutely and reversibly, in systemic or local fashion. These approaches make use of either drugs or genetically encoded tools. Also, there are acutely acting inhibitory tools that require an exogenous trigger like light. Here, we give an overview of such methods developed and used in the nematode Caenorhabditis elegans.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

The site-specific signal enhancement provided by parahydrogen induced polarization (PHIP) may be combined with magnetic resonance imaging (MRI) to study chemical and biomolecular processes. However, imaging of hydrogen nuclei (1H) is hampered by background signals arising from the presence of thermally polarized nuclei. Additionally, fast imaging sequences are commonly based on multiple radio-frequency pulses, where the signals resulting from PHIP oscillate due to the evolution with a J-coupling Hamiltonian. In this article, an innovative imaging scheme for single-scan MRI is presented that effectively detects hyperpolarized components while simultaneously canceling out thermal contributions. This method is based on the quenching of inherent oscillations of PHIP-originated signals due to J-couplings during the multipulse sequence and the suppression of thermal signals by spin dynamics and a tailored restructuring of the k-space. A series of numerical simulations on specific two- and three-spin systems serve to support the feasibility of the approach. Furthermore, this theoretical study demonstrates the potential of combining hyperpolarization and long-lived states (PHIP and LLS) in the selected molecules, which could be seen as a preliminary step towards the development of fast imaging techniques, for example in the field of biomolecular research.

OBJECTIVE: This study aimed to assess metabolic changes to monitor the progression from normal liver to hepatitis B virus (HBV)-related hepatitis and liver fibrosis using hyperpolarized 13C magnetic resonance imaging (MRI).
METHODS: Hepatitis was induced in mice (n = 16) via hydrodynamic injection of HBV 1.2 plasmid (25 μg). Among them, liver fibrosis was induced in the mice (n = 8) through weight-adapted administration of thioacetamide with ethanol. Normal control mice (n = 8) were injected with a phosphate buffer solution. Subsequently, a hyperpolarized 13C MRI was performed on the mouse liver in vivo. The level of hepatitis B surface antigen (HBsAg) in blood serum was measured. Statistical analysis involved comparing the differential metabolite ratios, blood biochemistry values, and body weight among the three groups using the Kruskal-Wallis one-way analysis of variance.
RESULTS: HBsAg was absent in the normal and fibrosis groups, while it was detected in the hepatitis group. The ratios of [1-13C] lactate/pyruvate, [1-13C] alanine/pyruvate, [1-13C] lactate/total carbon, and [1-13C] alanine/total carbon were significantly lower in the normal control group than in the hepatitis and fibrosis groups (p < 0.05). Moreover, these ratios were significantly higher in the fibrosis group than in the hepatitis group (p < 0.05). However, no significant differences were observed in either [1-13C] pyruvate-hydrate/pyruvate or [1-13C] pyruvate-hydrate/total carbon among the three groups.
CONCLUSIONS: The levels of [1-13C] lactate and [1-13C] alanine in vivo may serve as valuable indicators for differentiating between HBV-related hepatitis, liver fibrosis, and normal liver.