Detection of rare antigen-presenting cells through T cell-intrinsic meandering motility, mediated by Myo1g.

To mount an immune response, T lymphocytes must successfully search for foreign material bound to the surface of antigen-presenting cells. How T cells optimize their chances of encountering and responding to these antigens is unknown. T cell motility in tissues resembles a random or Levy walk and is regulated in part by external factors including chemokines and lymph-node topology, but motility parameters such as speed and propensity to turn may also be cell intrinsic. Here we found that the unconventional myosin 1g (Myo1g) motor generates membrane tension, enforces cell-intrinsic meandering search, and enhances T-DC interactions during lymph-node surveillance. Increased turning and meandering motility, as opposed to ballistic motility, is enhanced by Myo1g. Myo1g acts as a "turning motor" and generates a form of cellular "flânerie." Modeling and antigen challenges show that these intrinsically programmed elements of motility search are critical for the detection of rare cognate antigen-presenting cells.

Parametrically excited shape distortion of a submillimeter bubble.

The existence of finite amplitude shape distortion caused by parametrically excited surface instabilities for a gas bubble in water driven by a temporally periodic, spatially uniform pressure field in an axisymmetric geometry is investigated. Employing a nonlinear coupled system of equations which includes shape mode interactions to third order, the resultant spherical oscillations, translation, and shape distortion of the bubble are modelled, placing no restriction on the size of the spherical oscillations. The model accounts for viscous and thermal damping with compressibility effects. The existence of synchronous and higher order parametrically induced sustained, finite amplitude, periodic shape deformation is demonstrated. The excitement of an odd shape mode via the synchronous mechanism is shown to give rise to linear bubble self-propulsion. For larger driving amplitudes, it is shown that more than one shape mode can be parametrically excited at the same driving frequency but by different resonance mechanisms, leading to more involved shape deformation and the increased possibility of bubble self-propulsion.

Analyzing the impact of agricultural BMPs on stream nutrient load and biotic health in the Susquehanna-Chemung basin of New York.

Despite the widespread use of agricultural best management practices (BMPs) to reduce watershed scale nutrient loads, there remain few studies that use directly observed data - instead of models - to evaluate BMP effectiveness at the watershed scale. In this study, we make use of extensive ambient water quality data, stream biotic health data, and BMP implementation data within the New York State portion of the Chesapeake Bay watershed to assess the role of BMPs on reducing nutrient loads and modifying biotic health in major rivers. The specific BMPs considered were riparian buffers and nutrient management planning. A simple mass balance approach was used to evaluate the role of wastewater treatment plant nutrient reductions, agricultural land use changes, and these two agricultural BMPs in matching observed downward trends in nutrient load. In the Eastern nontidal network (NTN) catchment - where BMPs have been more widely reported - the mass balance model suggested a small but discernible contribution of BMPs in matching the observed downward trend in total phosphorus. Contrastingly, BMP implementations did not show clear contributions towards total nitrogen reductions in the Eastern NTN catchment nor for the total nitrogen and phosphorus in the Western NTN catchment, where BMP implementation data are more limited. Assessment of the relationship between stream biotic health and BMP implementation using regression models found limited connection between extent of BMP implementation and biotic health. In this case, however, spatiotemporal mismatches between the datasets and the relatively stable biotic health, typically of moderate to good quality even before BMP implementation, may reflect the need for better monitoring design to assess BMP effects at the subwatershed scale. Additional studies, perhaps using citizen scientists, may be able to provide more suitable data within the existing frameworks of the long-term surveys. Given the preponderance of studies that rely only on modeling to understand nutrient loading reductions achieved by implementation of BMPs, it is essential to continue to collect empirical data to meaningfully evaluate whether there are actual measurable changes due to BMPs.

BL-8040 CXCR4 antagonist is safe and demonstrates antileukemic activity in combination with cytarabine for the treatment of relapsed/refractory acute myelogenous leukemia: An open-label safety and efficacy phase 2a study.

BACKGROUND: CXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of the high-affinity CXCR4 antagonist BL-8040 with high-dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML. METHODS: Forty-two patients received treatment with BL-8040 monotherapy for 2 days followed by a combination of BL-8040 with HiDAC for 5 days. Six escalating BL-8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23). RESULTS: BL-8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL-8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5-mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5-mg/kg phase, and 21.8 months for responding patients in the 1.5-mg/kg cohort. Two days of BL-8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold-changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts. CONCLUSIONS: The current results demonstrate the efficacy of CXCR4 targeting with BL-8040 and support continued clinical development in acute myelogenous leukemia.

AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models.

BACKGROUND: Treatments that generate T cell-mediated immunity to a patient's unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galß1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. METHODS: Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT-/-) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel-Cox test, C5a data by Mann-Whitney test, and single tumor regression by repeated measures analysis. RESULTS: In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT-/- mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. CONCLUSIONS: We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

Environmental risk factors for canine atopic dermatitis: a retrospective large-scale study in Labrador and golden retrievers.

BACKGROUND: Canine atopic dermatitis (cAD) is one the most common and distressing skin disorders seen in dogs. It is characterized by dysfunction in the skin barrier, with a complex pathogenesis combining both genetic and environmental factors. OBJECTIVES: To evaluate associations between environmental factors and case-control status in two closely related, at-risk breeds, the Labrador retriever and golden retriever. ANIMALS: Two thousand four hundred and forty-five pet dogs, of which 793 were classed as cases (575 Labrador and 218 golden retrievers) and 1,652 as controls (1,120 Labrador and 532 golden retrievers). METHODS AND MATERIALS: Case-control status was assigned based upon owner response to a standardized validated questionnaire. Retrospective data on rearing environment were collected via additional questions. Univariate and multivariate logistic regressions were utilized to evaluate associations between environmental factors and case-control status. RESULTS: Risk factors included being reared in an urban environment (not living currently in an urban environment), being male, being neutered, receiving flea control and being allowed on upholstered furniture. Protective factors included living with other dogs (not cats) and walking in woodlands, fields or beaches. Additionally, amongst Labrador retrievers, chocolate-coloured dogs were at greater risk of having cAD than black- or yellow-coated dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: This study is the largest of its kind to date to investigate the role of the environment in cAD. Although precise triggers are unclear, this study complements earlier studies in highlighting the protective role of a rural environment and some novel associations with disease development.

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments.

Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice.

ERM (ezrin, radixin moesin) proteins in lymphocytes link cortical actin to plasma membrane, which is regulated in part by ERM protein phosphorylation. To assess whether phosphorylation of ERM proteins regulates lymphocyte migration and membrane tension, we generated transgenic mice whose T-lymphocytes express low levels of ezrin phosphomimetic protein (T567E). In these mice, T-cell number in lymph nodes was reduced by 27%. Lymphocyte migration rate in vitro and in vivo in lymph nodes decreased by 18% to 47%. Lymphocyte membrane tension increased by 71%. Investigations of other possible underlying mechanisms revealed impaired chemokine-induced shape change/lamellipod extension and increased integrin-mediated adhesion. Notably, lymphocyte homing to lymph nodes was decreased by 30%. Unlike most described homing defects, there was not impaired rolling or sticking to lymph node vascular endothelium but rather decreased migration across that endothelium. Moreover, decreased numbers of transgenic T cells in efferent lymph suggested defective egress. These studies confirm the critical role of ERM dephosphorylation in regulating lymphocyte migration and transmigration. Of particular note, they identify phospho-ERM as the first described regulator of lymphocyte membrane tension, whose increase probably contributes to the multiple defects observed in the ezrin T567E transgenic mice.

Antimicrobial resistance and characterisation of staphylococci isolated from healthy Labrador retrievers in the United Kingdom.

BACKGROUND: Coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci are normal commensals of the skin and mucosa, but are also opportunist pathogens. Meticillin-resistant (MR) and multidrug-resistant (MDR) isolates are increasing in human and veterinary healthcare. Healthy humans and other animals harbour a variety of staphylococci, including MR-CoPS and MR-CoNS. The main aims of the study were to characterise the population and antimicrobial resistance profiles of staphylococci from healthy non-vet visiting and non-antimicrobial treated Labrador retrievers in the UK. RESULTS: Nasal and perineal samples were collected from 73 Labrador retrievers; staphylococci isolated and identified using phenotypic and biochemical methods. They were also confirmed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), PCR of the nuc gene and PCR and sequencing of the tuf gene. Disc diffusion and minimum inhibitory concentration (MIC) susceptibility tests were determined for a range of antimicrobials. In total, 102 CoPS (S. pseudintermedius n = 91, S. aureus n = 11) and 334 CoNS isolates were detected from 99% of dogs in this study. In 52% of dogs CoNS only were detected, with both CoNS and CoPS detected in 43% dogs and CoPS only detected in 4% of dogs. Antimicrobial resistance was not common among CoPS, but at least one MDR-CoNS isolate was detected in 34% of dogs. MR-CoNS were detected from 42% of dogs but no MR-CoPS were isolated. S. epidermidis (52% of dogs) was the most common CoNS found followed by S. warneri (30%) and S. equorum (27%), with another 15 CoNS species isolated from ≤ 15% of dogs. S. pseudintermedius and S. aureus were detected in 44% and 8% of dogs respectively. CONCLUSIONS: MR- and MDR-CoPS were rare. However a high prevalence of MR- and MDR-CoNS were found in these dogs, even though they had no prior antimicrobial treatment or admission to veterinary premises. These findings are of concern due to the potential for opportunistic infections, zoonotic transmission and transmission of antimicrobial resistant determinants from these bacteria to coagulase positive staphylococci.

Improving risk estimates of runoff producing areas: formulating variable source areas as a bivariate process.

Predicting runoff producing areas and their corresponding risks of generating storm runoff is important for developing watershed management strategies to mitigate non-point source pollution. However, few methods for making these predictions have been proposed, especially operational approaches that would be useful in areas where variable source area (VSA) hydrology dominates storm runoff. The objective of this study is to develop a simple approach to estimate spatially-distributed risks of runoff production. By considering the development of overland flow as a bivariate process, we incorporated both rainfall and antecedent soil moisture conditions into a method for predicting VSAs based on the Natural Resource Conservation Service-Curve Number equation. We used base-flow immediately preceding storm events as an index of antecedent soil wetness status. Using nine sub-basins of the Upper Susquehanna River Basin, we demonstrated that our estimated runoff volumes and extent of VSAs agreed with observations. We further demonstrated a method for mapping these areas in a Geographic Information System using a Soil Topographic Index. The proposed methodology provides a new tool for watershed planners for quantifying runoff risks across watersheds, which can be used to target water quality protection strategies.

Enhanced effector function of CD8(+) T cells from healthy controls and HIV-infected patients occurs through thrombin activation of protease-activated receptor 1.

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.

Self-propulsion of a periodically forced shape-deforming submillimeter gas bubble.

The self-propulsion (translational instability) of a gas bubble in a liquid undergoing parametrically induced axisymmetric shape distortion due to being forced by a temporally sinusoidal, spatially constant acoustic field is investigated. Employing a model which accounts for the nonlinear coupling between the spherical oscillations, the axial translation and shape deformation of the bubble, the parametric excitement of two neighboring shape modes by the fundamental resonance, at the same driving frequency is studied. It is shown that provided pertinent driving pressure threshold values are exceeded, the respective shape modes are excited on different timescales. The growth of the shape mode on the faster timescale saturates giving rise to sustained constant amplitude oscillations, while the growth of the shape mode on the slower timescale is both modulated and unbounded. During the growth of the second shape mode, growing, oscillatory bubble translation is also observed.

Immunogenicity phase II study evaluating booster capacity of nonadjuvanted AKS-452 SARS-Cov-2 RBD Fc vaccine.

AKS-452, a subunit vaccine comprising an Fc fusion of the ancestral wild-type (WT) SARS-CoV-2 virus spike protein receptor binding domain (SP/RBD), was evaluated without adjuvant in a single cohort, non-randomized, open-labelled phase II study (NCT05124483) at a single site in The Netherlands for safety and immunogenicity. A single 90 µg subcutaneous booster dose of AKS-452 was administered to 71 adults previously primed with a registered mRNA- or adenovirus-based vaccine and evaluated for 273 days. All AEs were mild and no SAEs were attributable to AKS-452. While all subjects showed pre-existing SP/RBD binding and ACE2-inhibitory IgG titers, 60-68% responded to AKS-452 via ≥2-fold increase from days 28 to 90 and progressively decreased back to baseline by day 180 (days 28 and 90 mean fold-increases, 14.7 ± 6.3 and 8.0 ± 2.2). Similar response kinetics against RBD mutant proteins (including omicrons) were observed but with slightly reduced titers relative to WT. There was an expected strong inverse correlation between day-0 titers and the fold-increase in titers at day 28. AKS-452 enhanced neutralization potency against live virus, consistent with IgG titers. Nucleocapsid protein (Np) titers suggested infection occurred in 66% (46 of 70) of subjects, in which only 20 reported mild symptomatic COVID-19. These favorable safety and immunogenicity profiles support booster evaluation in a planned phase III universal booster study of this room-temperature stable vaccine that can be rapidly and inexpensively manufactured to serve vaccination at a global scale without the need of a complex distribution or cold chain.

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker.

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.

Myosin 1c participates in B cell cytoskeleton rearrangements, is recruited to the immunologic synapse, and contributes to antigen presentation.

Myosin 1c (Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma membrane with the microfilament cortical web. Although this molecular motor has been implicated in cell functions such as cytoskeleton organization, cell motility, nuclear transcription, and endocytosis, its role in hematopoietic cells is largely unknown. In this study, we show that Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located at the plasma membrane, especially in peripheral processes such as microvilli. We observed that this motor concentrates at the growing membrane protrusions generated during B cell spreading and that it is actively recruited to the immune synapse. Interestingly, Myo1c was detected in lipid rafts of B cells and showed strong colocalization with MHC-II, particularly after cross-linking of these molecules. By transfection of a dominant negative form of Myo1c or specific siRNA, we also detected alterations in the spreading and Ag-presenting ability of these cells. The data suggest that Myo1c is involved in the cytoskeleton dynamics and membrane protein anchoring or sorting in B lymphocytes.

Testing of a custom, portable drill press to minimize probe misalignment in sap flow sensors.

The accurate estimation of plant transpiration is critical to the fields of hydrology, plant physiology and ecology. Among the various methods of measuring transpiration in the field, the sap flow methods based on head pulses offers a cost-effective and energy-efficient option to directly measure the plant-level movement of water through the hydraulically active tissue. While authors have identified several possible sources of error in these measurements, one of the most common sources is misalignment of the sap flow probes due to user error. Though the effects of probe misalignment are well documented, no device or technique has been universally adopted to ensure the proper installation of sap flow probes. In this paper we compare the magnitude of misalignment errors among a 5 mm thick drilling template (DT), a 10 mm thick DT, and a custom designed, field-portable drill press. The different techniques were evaluated in the laboratory using a 7.5 cm wood block and in the field, comparing differences in measured sap flow. Based on analysis of holes drilled in the wood block, we found that the portable drill press was most effective in assuring that drill holes remained parallel, even at 7.5 cm depth. In field installations, nearly 50% of holes drilled with a 5 mm template needed to be redrilled while none needed to be when drilled with the drill press. Widespread use of a portable drill press when implementing the heat pulse method would minimize alignment uncertainty and allow a clearer understanding of other sources of uncertainty due to variability in tree species, age, or external drivers or transpiration.

Pseudomonas spp. in Canine Otitis Externa.

Canine otitis externa (OE) is a commonly diagnosed condition seen in veterinary practice worldwide. In this review, we discuss the mechanisms of the disease, with a particular focus on the biological characteristics of Pseudomonas aeruginosa and the impact that antibiotic resistance has on successful recovery from OE. We also consider potential alternatives to antimicrobial chemotherapy for the treatment of recalcitrant infections. P. aeruginosa is not a typical constituent of the canine ear microbiota, but is frequently isolated from cases of chronic OE, and the nature of this pathogen often makes treatment difficult. Biofilm formation is identified in 40-95% of P. aeruginosa from cases of OE and intrinsic and acquired antibiotic resistance, especially resistance to clinically important antibiotics, highlights the need for alternative treatments. The role of other virulence factors in OE remains relatively unexplored and further work is needed. The studies described in this work highlight several potential alternative treatments, including the use of bacteriophages. This review provides a summary of the aetiology of OE with particular reference to the dysbiosis that leads to colonisation by P. aeruginosa and highlights the need for novel treatments for the future management of P. aeruginosa otitis.

Small-molecule inhibitors of the LEDGF/p75 binding site of integrase block HIV replication and modulate integrase multimerization.

Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.

Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.

The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4(+) T cells exhibit decreased protein kinase C (PKC)-theta recruitment upon TCR stimulation. We demonstrate in this paper using GST pulldowns and coimmunoprecipitation studies that SAP constitutively associates with PKC- in T cells. SAP-PKC-theta interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP's interactions with PKC-theta occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-theta recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-theta, like SAP, was required for SLAM-mediated increases in IL-4 production, and, conversely, membrane-targeted PKC-theta mutants rescued IL-4 expression in SAP(-/-) CD4(+) T cells, providing genetic evidence that PKC-theta is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.