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Fluorinated, cell-permeable analogs of sialic acid and fucose are processed by monosaccharide salvage pathways to generate sialyl- and fucosyltransferase inhibitors intracellularly. These compounds serve as important new tools to dissect the role of glycan modifications within complex biological systems. Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate–based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimeric integrin consisting of α(L) (gene name, Itgal) and β(2) (gene name, Itgb2) subunits expressed in all leukocytes. LFA-1 is essential for neutrophil recruitment to inflamed tissue. Activation of LFA-1 by chemokines allows neutrophils and other leukocytes to undergo arrest, resulting in firm adhesion on endothelia expressing intercellular adhesion molecules (ICAMs). In mice, CXCR2 is the primary chemokine receptor involved in triggering neutrophil arrest, and it does so through \"inside-out\" activation of LFA-1. CXCR2 signaling induces changes in LFA-1 conformation that are coupled to affinity upregulation of the ligand-binding headpiece (extended with open I domain). Unlike naïve lymphocytes, engagement of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils stimulates a slow rolling behavior that is mediated by LFA-1 in a distinct activation state (extended with closed I domain). How inside-out signaling cascades regulate the structure and function of LFA-1 is being studied using flow chambers, intravital microscopy, and flow cytometry for ligand and reporter antibody binding. Here, we review how LFA-1 activation is regulated by cellular signaling and ligand binding. Two FERM domain-containing proteins, talin-1 and Kindlin-3, are critical integrin co-activators and have distinct roles in the induction of LFA-1 conformational rearrangements. This review integrates these new results into existing models of LFA-1 activation.

Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of Staphylococcus aureus by neutrophils can be monitored using a S. aureus strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent S. aureus killing assay for use in vivo. Conjugating S. aureus with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed S. aureus , and cells that have viable internalized S. aureus . We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent S. aureus due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the in vivo utility of ATTO647N/GFP bi-fluorescent S. aureus , we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of S. aureus infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent S. aureus demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.

High-precision searches for an electric dipole moment of the neutron (nEDM) require stable and uniform magnetic field environments. We present the recent achievements of degaussing and equilibrating the magnetically shielded room (MSR) for the n2EDM experiment at the Paul Scherrer Institute. We present the final degaussing configuration that will be used for n2EDM after numerous studies. The optimized procedure results in a residual magnetic field that has been reduced by a factor of two. The ultra-low field is achieved with the full magnetic-field-coil system, and a large vacuum vessel installed, both in the MSR. In the inner volume of ∼ 1.4 m 3 , the field is now more uniform and below 300 pT. In addition, the procedure is faster and dissipates less heat into the magnetic environment, which in turn, reduces its thermal relaxation time from 12 h down to 1.5 h .

We present a novel Active Magnetic Shield (AMS), designed and implemented for the n2EDM experiment at the Paul Scherrer Institute. The experiment will perform a high-sensitivity search for the electric dipole moment of the neutron. Magnetic-field stability and control is of key importance for n2EDM. A large, cubic, 5 m side length, magnetically shielded room (MSR) provides a passive, quasi-static shielding-factor of about [Formula omitted] for its inner sensitive volume. The AMS consists of a system of eight complex, feedback-controlled compensation coils constructed on an irregular grid spanned on a volume of less than 1000 m [Formula omitted] around the MSR. The AMS is designed to provide a stable and uniform magnetic-field environment around the MSR, while being reasonably compact. The system can compensate static and variable magnetic fields up to [Formula omitted] (homogeneous components) and [Formula omitted] (first-order gradients), suppressing them to a few [Formula omitted] in the sub-Hertz frequency range. The presented design concept and implementation of the AMS fulfills the requirements of the n2EDM experiment and can be useful for other applications, where magnetically silent environments are important and spatial constraints inhibit simpler geometrical solutions.

We report on a search for a new, short-range, spin-dependent interaction using a modified version of the experimental apparatus used to measure the permanent neutron electric dipole moment at the Paul Scherrer Institute. This interaction, which could be mediated by axion-like particles, concerned the unpolarized nucleons (protons and neutrons) near the material surfaces of the apparatus and polarized ultracold neutrons stored in vacuum. The dominant systematic uncertainty resulting from magnetic-field gradients was controlled to an unprecedented level of approximately 4 pT cm −1 using an array of optically-pumped cesium vapor magnetometers and magnetic-field maps independently recorded using a dedicated measurement device. No signature of a theoretically predicted new interaction was found, and we set a new limit on the product of the scalar and the pseudoscalar couplings g s g p λ 2 < 8.3 × 10 − 28 m 2 (95% C.L.) in a range of 5  µ m < λ < 25  mm for the monopole–dipole interaction. This new result confirms and improves our previous limit by a factor of 2.7 and provides the current tightest limit obtained with free neutrons.

The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to the in vivo setting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.

We present the design of a next-generation experiment, n2EDM, currently under construction at the ultracold neutron source at the Paul Scherrer Institute (PSI) with the aim of carrying out a high-precision search for an electric dipole moment of the neutron. The project builds on experience gained with the previous apparatus operated at PSI until 2017, and is expected to deliver an order of magnitude better sensitivity with provision for further substantial improvements. An overview is of the experimental method and setup is given, the sensitivity requirements for the apparatus are derived, and its technical design is described.

Bacterial community activity and structure are thought to be directly or indirectly related to phytoplankton development and, in particular, to the phytoplankton species dominating specific algal blooms. To test this hypothesis, we performed a mesocosm experiment designed to generate blooms of different types of phytoplankton through the additions of silicate, urea and phosphorus to oligotrophic water from the Blanes Bay Microbial Observatory (NW Mediterranean). Over 10 d of incubation bacterial activity, bacterial abundance, nutrient composition and free-living bacterial community structure were monitored, as well as phytoplankton composition and the fluorescence characteristics of dissolved organic matter (DOM). While we found clear effects of the different nutrient additions on chlorophyll levels, bacterial production and the type of dominant DOM, bacterial abundance followed a similar pattern across different nutrient treatments, which deviated from that observed in the control. While phytoplankton composition in the treatment with added silicate evolved differently with respect to the other treatments, free-living bacterial community structure (as determined with DGGE) did not show conspicuous differences between treatments. Our results reveal that the changes in bacterial community composition were mostly due to the variation in grazing pressure with time, with a small contribution from changes in bottom-up nutrient supply mediated by the shifts in phytoplankton composition.

Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in β2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. However, we also found that vinculin deficiency does not affect these behaviors in the presence of forces from fluid flow. In addition, we identify a role for vinculin in mechanosensing, as vinculin-deficient neutrophils exhibit attenuated spreading on stiff, but not soft, substrates. Consistent with these findings, we observe that in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for β2 integrin-dependent neutrophil recruitment in vivo .