Realizing spin squeezing with Rydberg interactions in an optical clock.

Neutral-atom arrays trapped in optical potentials are a powerful platform for studying quantum physics, combining precise single-particle control and detection with a range of tunable entangling interactions1-3. For example, these capabilities have been leveraged for state-of-the-art frequency metrology4,5 as well as microscopic studies of entangled many-particle states6-11. Here we combine these applications to realize spin squeezing-a widely studied operation for producing metrologically useful entanglement-in an optical atomic clock based on a programmable array of interacting optical qubits. In this demonstration of Rydberg-mediated squeezing with a neutral-atom optical clock, we generate states that have almost four decibels of metrological gain. In addition, we perform a synchronous frequency comparison between independent squeezed states and observe a fractional-frequency stability of 1.087(1) × 10-15 at one-second averaging time, which is 1.94(1) decibels below the standard quantum limit and reaches a fractional precision at the 10-17 level during a half-hour measurement. We further leverage the programmable control afforded by optical tweezer arrays to apply local phase shifts to explore spin squeezing in measurements that operate beyond the relative coherence time with the optical local oscillator. The realization of this spin-squeezing protocol in a programmable atom-array clock will enable a wide range of quantum-information-inspired techniques for optimal phase estimation and Heisenberg-limited optical atomic clocks12-16.

Resolving the gravitational redshift across a millimetre-scale atomic sample.

Einstein's theory of general relativity states that clocks at different gravitational potentials tick at different rates relative to lab coordinates-an effect known as the gravitational redshift1. As fundamental probes of space and time, atomic clocks have long served to test this prediction at distance scales from 30 centimetres to thousands of kilometres2-4. Ultimately, clocks will enable the study of the union of general relativity and quantum mechanics once they become sensitive to the finite wavefunction of quantum objects oscillating in curved space-time. Towards this regime, we measure a linear frequency gradient consistent with the gravitational redshift within a single millimetre-scale sample of ultracold strontium. Our result is enabled by improving the fractional frequency measurement uncertainty by more than a factor of 10, now reaching 7.6 × 10-21. This heralds a new regime of clock operation necessitating intra-sample corrections for gravitational perturbations.

Stabilin receptors clear LPS and control systemic inflammation.

Lipopolysaccharides (LPSs) cause lethal endotoxemia if not rapidly cleared from blood circulation. Liver sinusoidal endothelial cells (LSEC) systemically clear LPS by unknown mechanisms. We discovered that LPS clearance through LSEC involves endocytosis and lysosomal inactivation via Stabilin-1 and 2 (Stab1 and Stab2) but does not involve TLR4. Cytokine production was inversely related to clearance/endocytosis of LPS by LSEC. When exposed to LPS, Stabilin double knockout mice (Stab DK) and Stab1 KO, but not Stab2 KO, showed significantly enhanced systemic inflammatory cytokine production and early death compared with WT mice. Stab1 KO is not significantly different from Stab DK in circulatory LPS clearance, LPS uptake and endocytosis by LSEC, and cytokine production. These data indicate that (1) Stab1 receptor primarily facilitates the proactive clearance of LPS and limits TLR4-mediated inflammation and (2) TLR4 and Stab1 are functionally opposing LPS receptors. These findings suggest that endotoxemia can be controlled by optimizing LPS clearance by Stab1.

Thermal noise and mechanical loss of SiO2/Ta2O5 optical coatings at cryogenic temperatures.

Mechanical loss of dielectric mirror coatings sets fundamental limits for both gravitational wave detectors and cavity-stabilized optical local oscillators for atomic clocks. Two approaches are used to determine the mechanical loss: ringdown measurements of the coating quality factor and direct measurement of the coating thermal noise. Here we report a systematic study of the mirror thermal noise at 4, 16, 124, and 300 K by operating reference cavities at these temperatures. The directly measured thermal noise is used to extract the mechanical loss for SiO2/Ta2O5 coatings, which are compared with previously reported values.

Accelerated Clearance and Degradation of Cell-Free HIV by Neutralizing Antibodies Occurs via FcγRIIb on Liver Sinusoidal Endothelial Cells by Endocytosis.

Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy.

Precision Metrology Meets Cosmology: Improved Constraints on Ultralight Dark Matter from Atom-Cavity Frequency Comparisons.

We conduct frequency comparisons between a state-of-the-art strontium optical lattice clock, a cryogenic crystalline silicon cavity, and a hydrogen maser to set new bounds on the coupling of ultralight dark matter to standard model particles and fields in the mass range of 10^{-16}-10^{-21} eV. The key advantage of this two-part ratio comparison is the differential sensitivity to time variation of both the fine-structure constant and the electron mass, achieving a substantially improved limit on the moduli of ultralight dark matter, particularly at higher masses than typical atomic spectroscopic results. Furthermore, we demonstrate an extension of the search range to even higher masses by use of dynamical decoupling techniques. These results highlight the importance of using the best-performing atomic clocks for fundamental physics applications, as all-optical timescales are increasingly integrated with, and will eventually supplant, existing microwave timescales.

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca2+ and to eliminate one of the inactive states at low Ca2+. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca2+, full activation with Δ14-TnT·A8V-TnC decreased the apparent KM for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca2+ with Δ14-TnT; the maximum shortening speed at high Ca2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca2+, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

Cardiac troponin and tropomyosin bind to F-actin cooperatively, as revealed by fluorescence microscopy.

In cardiac muscle, binding of troponin (Tn) and tropomyosin (Tpm) to filamentous (F)-actin forms thin filaments capable of Ca2+ -dependent regulation of contraction. Tpm binds to F-actin in a head-to-tail fashion, while Tn stabilizes these linkages. Valuable structural and functional information has come from biochemical, X-ray, and electron microscopy data. However, the use of fluorescence microscopy to study thin filament assembly remains relatively underdeveloped. Here, triple fluorescent labeling of Tn, Tpm, and F-actin allowed us to track thin filament assembly by fluorescence microscopy. It is shown here that Tn and Tpm molecules self-organize on actin filaments and give rise to decorated and undecorated regions. Binding curves based on colocalization of Tn and Tpm on F-actin exhibit cooperative binding with a dissociation constant Kd of ~ 0.5 µm that is independent of the Ca2+ concentration. Binding isotherms based on the intensity profile of fluorescently labeled Tn and Tpm on F-actin show that binding of Tn is less cooperative relative to Tpm. Computational modeling of Tn-Tpm binding to F-actin suggests two equilibrium steps involving the binding of an initial Tn-Tpm unit (nucleation) and subsequent recruitment of adjacent Tn-Tpm units (elongation) that stabilize the assembly. The results presented here highlight the utility of employing fluorescence microscopy to study supramolecular protein assemblies.

Demonstration of a Timescale Based on a Stable Optical Carrier.

We report on the first timescale based entirely on optical technology. Existing timescales, including those incorporating optical frequency standards, rely exclusively on microwave local oscillators owing to the lack of an optical oscillator with the required frequency predictability and stability for reliable steering. We combine a cryogenic silicon cavity exhibiting improved long-term stability and an accurate ^{87}Sr lattice clock to form a timescale that outperforms them all. Our timescale accumulates an estimated time error of only 48±94 ps over 34 days of operation. Our analysis indicates that this timescale is capable of reaching a stability below 1×10^{-17} after a few months of averaging, making timekeeping at the 10^{-18} level a realistic prospect.

Ca2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca2+ binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca2+ binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca2+ interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca2+- and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca2+ was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca2+ conditions but not to the degree seen in thin filaments with Ca2+. ATP-γ-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca2+ levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca2+ and the myosin catalytic cycle state.

T-cell target antigens across major gynecologic cancers.

Immunotherapies have achieved remarkable success in treating different forms of cancer including melanoma, non-small cell lung carcinoma, bladder cancer, synovial cell sarcoma, and multiple myeloma using immune checkpoint blockade or gene-engineered T-cells. Although gynecologic cancers have not been historically classified as immunogenic tumors, growing evidence has shown that they are in fact able to elicit endogenous antitumor immune responses suggesting that patients with these cancers may benefit from immunotherapy. Modest clinical success has been accomplished in early trials using immunotherapeutic modalities for major gynecologic cancers including ovarian, cervical, and endometrial cancer. Unlike solid cancers with high mutational burdens, or hematologic malignancies where target antigens are expressed homogenously and exclusively by tumor cells, identifying tumor-restricted antigens has been challenging when designing a T-cell targeted therapy for gynecologic tumors. Nevertheless, mounting preclinical and clinical evidence suggests that targeting shared, viral or patient-specific mutated antigens expressed by gynecologic tumors with T-cells may improve patient outcome. Here we review the strengths and weaknesses of targeting these various antigens, as well as provide insight into the future of immunotherapy for gynecologic cancers.

Mouse Liver Sinusoidal Endothelium Eliminates HIV-Like Particles from Blood at a Rate of 100 Million per Minute by a Second-Order Kinetic Process.

We crafted human immunodeficiency virus (HIV)-like particles of diameter about 140 nm, which expressed two major HIV-1 proteins, namely, env and gag gene products, and used this reagent to simulate the rate of decay of HIV from the blood stream of BALB/c male mice. We found that most (~90%) of the particles were eliminated (cleared) from the blood by the liver sinusoidal endothelial cells (LSECs), the remainder from Kupffer cells; suggesting that LSECs are the major liver scavengers for HIV clearance from blood. Decay was rapid with kinetics suggesting second order with respect to particles, which infers dimerization of a putative receptor on LSEC. The number of HIV-like particles required for saturating the clearance mechanism was approximated. The capacity for elimination of blood-borne HIV-like particles by the sinusoid was 112 million particles per minute. Assuming that the sinusoid endothelial cells were about the size of glass-adherent macrophages, then elimination capacity was more than 540 particles per hour per endothelial cell.

Blood-Borne Lipopolysaccharide Is Rapidly Eliminated by Liver Sinusoidal Endothelial Cells via High-Density Lipoprotein.

During Gram-negative bacterial infections, excessive LPS induces inflammation and sepsis via action on immune cells. However, the bulk of LPS can be cleared from circulation by the liver. Liver clearance is thought to be a slow process mediated exclusively by phagocytic resident macrophages, Kupffer cells (KC). However, we discovered that LPS disappears rapidly from the circulation, with a half-life of 2-4 min in mice, and liver eliminates about three quarters of LPS from blood circulation. Using microscopic techniques, we found that ∼75% of fluor-tagged LPS in liver became associated with liver sinusoidal endothelial cells (LSEC) and only ∼25% with KC. Notably, the ratio of LSEC-KC-associated LPS remained unchanged 45 min after infusion, indicating that LSEC independently processes the LPS. Most interestingly, results of kinetic analysis of LPS bioactivity, using modified limulus amebocyte lysate assay, suggest that recombinant factor C, an LPS binding protein, competitively inhibits high-density lipoprotein (HDL)-mediated LPS association with LSEC early in the process. Supporting the previous notion, 3 min postinfusion, 75% of infused fluorescently tagged LPS-HDL complex associates with LSEC, suggesting that HDL facilitates LPS clearance. These results lead us to propose a new paradigm of LSEC and HDL in clearing LPS with a potential to avoid inflammation during sepsis.

Wafer-scale monodomain films of spontaneously aligned single-walled carbon nanotubes.

The one-dimensional character of electrons, phonons and excitons in individual single-walled carbon nanotubes leads to extremely anisotropic electronic, thermal and optical properties. However, despite significant efforts to develop ways to produce large-scale architectures of aligned nanotubes, macroscopic manifestations of such properties remain limited. Here, we show that large (>cm(2)) monodomain films of aligned single-walled carbon nanotubes can be prepared using slow vacuum filtration. The produced films are globally aligned within ±1.5° (a nematic order parameter of ∼1) and are highly packed, containing 1 × 10(6) nanotubes in a cross-sectional area of 1 µm(2). The method works for nanotubes synthesized by various methods, and film thickness is controllable from a few nanometres to ∼100 nm. We use the approach to create ideal polarizers in the terahertz frequency range and, by combining the method with recently developed sorting techniques, highly aligned and chirality-enriched nanotube thin-film devices. Semiconductor-enriched devices exhibit polarized light emission and polarization-dependent photocurrent, as well as anisotropic conductivities and transistor action with high on/off ratios.

Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells.

Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with ß-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.

FluoroNanogold: an important probe for correlative microscopy.

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.

Digital microfluidic platform for the detection of rubella infection and immunity: a proof of concept.

BACKGROUND: Whereas disease surveillance for infectious diseases such as rubella is important, it is critical to identify pregnant women at risk of passing rubella to their offspring, which can be fatal and can result in congenital rubella syndrome (CRS). The traditional centralized model for diagnosing rubella is cost-prohibitive in resource-limited settings, representing a major obstacle to the prevention of CRS. As a step toward decentralized diagnostic systems, we developed a proof-of-concept digital microfluidic (DMF) diagnostic platform that possesses the flexibility and performance of automated immunoassay platforms used in central facilities, but with a form factor the size of a shoebox. METHODS: DMF immunoassays were developed with integrated sample preparation for the detection of rubella virus (RV) IgG and IgM. The performance (sensitivity and specificity) of the assays was evaluated with serum and plasma samples from a commercial antirubella mixed-titer performance panel. RESULTS: The new platform performed the essential processing steps, including sample aliquoting for 4 parallel assays, sample dilution, and IgG blocking. Testing of performance panel samples yielded diagnostic sensitivity and specificity of 100% and 100% for both RV IgG and RV IgM. With 1.8 µL sample per assay, 4 parallel assays were performed in approximately 30 min with <10% mean CV. CONCLUSIONS: This proof of concept establishes DMF-powered immunoassays as being potentially useful for the diagnosis of infectious disease.

Cell fusion mediates dramatic alterations in the actin cytoskeleton, focal adhesions, and E-cadherin in trophoblastic cells.

The syncytiotrophoblast of the human placenta is a unique epithelia structure with millions of nuclei sharing a common cytoplasm. The syncytiotrophoblast forms by cell-cell fusion of cytotrophoblasts (CTB), the mononuclear precursor cells. The trophoblastic BeWo cell line has been used as a surrogate for CTB since they can be induced to fuse, and subsequently display numerous syncytiotrophoblast differentiation markers following syncytial formation. In this study, we have focused on alterations in the cell-adhesion molecule E-cadherin, actin cytoskeleton, and focal adhesions following BeWo cell fusion, since these entities may be interrelated. There was a dramatic reorganization of the distribution of E-cadherin as well as a reduction in the amount of E-cadherin following cell fusion. Reorganization of the actin cytoskeleton was also observed, which was associated with a change in the globular actin (G-actin)/filamentous actin (F-actin) ratio. Concomitantly, the morphology of focal adhesions was altered, but this occurred without a corresponding change in the levels of focal adhesion marker proteins. Thus, extensive remodeling of the actin cytoskeleton and focal adhesions accompanies cell fusion and differentiation and appears related to alterations in E-cadherin in trophoblastic cells.

Determinants of pore folding in potassium channel biogenesis.

Many ion channels, both selective and nonselective, have reentrant pore loops that contribute to the architecture of the permeation pathway. It is a fundamental feature of these diverse channels, regardless of whether they are gated by changes of membrane potential or by neurotransmitters, and is critical to function of the channel. Misfolding of the pore loop leads to loss of trafficking and expression of these channels on the cell surface. Mature tetrameric potassium channels contain an α-helix within the pore loop. We systematically mutated the "pore helix" residues of the channel Kv1.3 and assessed the ability of the monomer to fold into a tertiary reentrant loop. Our results show that pore loop residues form a canonical α-helix in the monomer early in biogenesis and that disruption of tertiary folding is caused by hydrophilic substitutions only along one face of this α-helix. These results provide insight into the determinants of the reentrant pore conformation, which is essential for ion channel function.