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Valvular heart disease (VHD) leading to inadequate hemodynamic circulation is a major cause of cardiovascular morbidity and mortality worldwide. Right ventricular-pulmonary artery (RV–PA) coupling integrates the ability of RV contractility to adapt to increased pulmonary arterial afterload. If the right ventricle cannot adapt to the elevated afterload by increasing its contractile function, RV-PA uncoupling occurs. RV-PA uncoupling has been shown to be associated with poor outcomes in VHD. This review summarizes the prognostic significance of RV-PA coupling in patients with VHD.

The integration of a photodetector that converts optical signals into electrical signals is essential for scalable integrated lithium niobate photonics. Two-dimensional materials provide a potential high-efficiency on-chip detection capability. Here, we demonstrate an efficient on-chip photodetector based on a few layers of MoTe2 on a thin film lithium niobate waveguide and integrate it with a microresonator operating in an optical telecommunication band. The lithium-niobate-on-insulator waveguides and micro-ring resonator are fabricated using the femtosecond laser photolithography-assisted chemical–mechanical etching method. The lithium niobate waveguide-integrated MoTe2 presents an absorption coefficient of 72% and a transmission loss of 0.27 dB µm−1 at 1550 nm. The on-chip photodetector exhibits a responsivity of 1 mA W−1 at a bias voltage of 20 V, a low dark current of 1.6 nA, and a photo–dark current ratio of 108 W−1. Due to effective waveguide coupling and interaction with MoTe2, the generated photocurrent is approximately 160 times higher than that of free-space light irradiation. Furthermore, we demonstrate a wavelength-selective photonic device by integrating the photodetector and micro-ring resonator with a quality factor of 104 on the same chip, suggesting potential applications in the field of on-chip spectrometers and biosensors.

We demonstrate high-extraction-rate Ta2O5-core/SiO2-clad photonic waveguides on silicon fabricated by the photolithography-assisted chemo-mechanical etching technique. Low-confinement waveguides of larger than 70% coupling efficiency with optical fibers and medium propagation loss around 1 dB/cm are investigated in the experiment. Monolithic microring resonators based on Ta2O5 waveguides have shown the quality factors to be above 105 near 1550 nm. The demonstrated Ta2O5 waveguides and their fabrication method hold great promise in various cost-effective applications, such as optical interconnecting and switching.

Introduction and Objectives: Cholangiocarcinoma (CCA) is characterized by early distant invasion and metastasis, whereas the underlying mechanism is still obscure. Increasing evidence shows that collagen type Ι alpha 1 (COL1A1) is a gene associated with the progression of multiple diseases. Here, we attempted to investigate the role of COL1A1 in CCA. Materials and Methods: The expression of COL1A1 between tumor tissues and adjacent normal tissues obtained from CCA patients was detected by Western blot and immunofluorescence, followed by analysis of its clinical significance. Then, the biological effects of COL1A1 overexpression or knockdown on CCA cells were evaluated in vitro and in vivo. Finally, molecular mechanism of COL1A1 in regulating the invasion and metastasis of CCA cells was determined by a series of experiments. Results: COL1A1 expression was significantly higher in CCA pathological tissues than in corresponding adjacent normal tissues. Analysis of 83 CCA patients showed that higher expression of COL1A1 was correlated with poorer patient prognosis. Notably, overexpression or knockdown experiments revealed that COL1A1 contributed to the migration and invasion, as well as epithelial-to-mesenchymal transition (EMT), in CCA cells. Further investigations demonstrated that matrix metalloproteinase-2 (MMP2) promoted COL1A1 upregulation via the integrin alpha Ⅴ pathway, therefore affecting ECM remodelling and inducing EMT in CCA cells. Moreover, COL1A1 expression was positively related to PD-1 and PD-L1 in CCA, and COL1A1 increased PD-L1 expression by activating the NF-κB pathway. Conclusions: COL1A1 plays an important role in regulating CCA progression and may act as a promising biomarker and therapeutic target for CCA.

Abstract Background Biliary complications, especially non‐anastomotic stricture (NAS), are the main complications after liver transplantation. Insufficient sampling and no recognized animal models obstruct the investigation. Thus, the mechanisms and alterations that occur during endoscopic treatment (ET) of NAS remain unclear. Methods Samples were obtained with endoscopic forceps from the hilar bile ducts of NAS patients receiving continuous biliary stent implantation after diagnosis. Retrospective analysis of multiple studies indicated that the duration of ET for NAS was approximately 1–2 years. Thus, we divided the patients into short‐term treatment (STT) and long‐term treatment (LTT) groups based on durations of less or more than 1 year. Samples were subjected to single‐cell RNA sequencing. Transcriptomic differences between STT and normal groups were defined as the NAS mechanism. Similarly, alterations from STT to LTT groups were regarded as endoscopic‐treatment‐induced evolution. Results In NAS, inflammation and immune‐related pathways were upregulated in different cell types, with nonimmune cells showing hypoxia pathway upregulation and immune cells showing ATP metabolism pathway upregulation, indicating heterogeneity. We confirmed a reduction in bile acid metabolism‐related SPP1+ epithelial cells in NAS. Increases in proinflammatory and profibrotic fibroblast subclusters indicated fibrotic progression in NAS. Furthermore, immune disorders in NAS were exacerbated by an increase in plasma cells and dysfunction of NK and NKT cells. ET downregulated multicellular immune and inflammatory responses and restored epithelial and endothelial cell proportions. Conclusions This study reveals the pathophysiological and genetic mechanisms and evolution of NAS induced by ET, thereby providing preventive and therapeutic insights into NAS. Highlights For the first time, single‐cell transcriptome sequencing was performed on the bile ducts of patients with biliary complications. scRNA‐seq analysis revealed distinct changes in the proportion and phenotype of multiple cell types during Nonanastomotic stricture (NAS) and endoscopic treatment. A reduction in bile acid metabolism‐related SPP1+ epithelial cells and VEGFA+ endothelial cells, along with explosive infiltration of plasma cells and dysfunction of T and NK cells in NAS patients. SPP1+ macrophages and BST2+ T cells might serve as a surrogate marker for predicting endoscopic treatment.

Semantic segmentation, which is a fundamental task in computer vision. Every pixel will have a specific semantic class assigned to it through semantic segmentation methods. Embedded systems and mobile devices are difficult to deploy high-accuracy segmentation algorithms. Despite the rapid development of semantic segmentation, the balance between speed and accuracy must be improved. As a solution to the above problems, we created a cross-scale fusion attention mechanism network called CFANet, which fuses feature maps from different scales. We first design a novel efficient residual module (ERM), which applies both dilation convolution and factorized convolution. Our CFANet is mainly constructed from ERM. Subsequently, we designed a new multi-branch channel attention mechanism (MCAM) to refine the feature maps at different levels. Experiment results show that CFANet achieved 70.6% mean intersection over union (mIoU) and 67.7% mIoU on Cityscapes and CamVid datasets, respectively, with inference speeds of 118 FPS and 105 FPS on NVIDIA RTX2080Ti GPU cards with 0.84M parameters.

Pulmonary regurgitation usually leads to right heart dilatation and eventually right heart dysfunction, which is associated with a poor prognosis. Transcatheter pulmonary valve replacement is a developing treatment for pulmonary valve dysfunction that can take the place of traditional surgery and make up for the shortcomings of a large injury. Echocardiography plays a significant role in assessing ventricular function; however, conventional echocardiographic parameters have several limitations. Speckle tracking echocardiography has been regarded as a more accurate tool for quantifying cardiac function than conventional echocardiography. Therefore, the aim of this review was to summarize the application of speckle tracking echocardiography for evaluating right and left ventricular functions in patients after transcatheter pulmonary valve replacement.

Heart failure with preserved ejection fraction (HFpEF) is defined as HF with left ventricular ejection fraction (LVEF) not less than 50%. HFpEF accounts for more than 50% of all HF patients, and its prevalence is increasing year to year with the aging population, with its prognosis worsening. The clinical assessment of cardiac function and prognosis in patients with HFpEF remains challenging due to the normal range of LVEF and the nonspecific symptoms and signs. In recent years, new echocardiographic techniques have been continuously developed, particularly speckle-tracking echocardiography (STE), which provides a sensitive and accurate method for the comprehensive assessment of cardiac function and prognosis in patients with HFpEF. Therefore, this article reviewed the clinical utility of STE in patients with HFpEF.

ObjectivesBoth right ventricular free wall longitudinal strain (RVFWLS) and right ventricular global longitudinal strain (RVGLS) using two-dimensional speckle tracking echocardiography (2D-STE) has been demonstrated to predict adverse outcomes in patients with repaired tetralogy of Fallot (r-TOF). However, RVGLS may be affected by left ventricular (LV) function owing to the fact that the interventricular septum is also a part of the left ventricle. Therefore, the aim of our study was to compare the predictive value of RVFWLS with that of RVGLS in patients with r-TOF.Materials and methodsA total of 179 patients with r-TOF were included in this study. RVFWLS, RVGLS, and left ventricle global longitudinal strain (LVGLS) were evaluated by 2D-STE. The adverse clinical events were death or r-TOF-related rehospitalization. Prognostic performance was evaluated by C-statistic and Akaike information criterion (AIC).ResultsThirty-one patients developed poor outcomes during a median follow-up period of 2.8 years. Compared with patients without end-point events, those with end-point events had higher incidence of moderate/severe pulmonary regurgitation, larger right heart sizes, and lower RV fractional area change (RVFAC), RVFWLS, RVGLS, and LVGLS than those without. Multivariate Cox regression analysis revealed that RVFAC, RVFWLS, RVGLS, and LVGLS were predictive of poor outcomes in patients with r-TOF after adjustment for transannular patch and QRS duration. A Cox model using RVFWLS (C index = 0.876, AIC = 228) was found to predict unfavorable outcomes more accurately than a model with RVGLS (C index = 0.856, AIC = 243), RVFAC (C index = 0.811, AIC = 248), and LVGLS (C index = 0.830, AIC = 248).ConclusionAlthough both RVGLS and RVFWLS are associated with adverse events, RVFWLS provides superior prognostic value than that of RVGLS in patients with r-TOF.

BackgroundAlthough the left atrium (LA) plays a key role in the pathophysiology and disease progression of heart failure with preserved ejection fraction (HFpEF), the impact of type 2 diabetes mellitus (T2DM) on LA function and stiffness in HFpEF patients remains unclear. Furthermore, the prognostic value of different phases of LA function and stiffness is less well-established in HFpEF patients.MethodsThis study prospectively enrolled 164 HFpEF patients who were in sinus rhythm at the time of echocardiography, including 61 (37%) HFpEF patients with T2DM. LA reservoir, conduit, and pump function were assessed using two-dimensional volume indices and speckle tracking echocardiography. The LA stiffness was calculated as the ratio of early mitral inflow velocity-to-early annular tissue velocity (E/e’) and LA reservoir function. The primary end point was a combined outcome of heart failure hospitalization or death.ResultsLeft atrium reservoir function [measured by peak LA strain (LAS-peak)] and LA pump function (measured by LAS-active) remained significantly lower in the HFpEF patients with T2DM compared with those without T2DM, even after adjustment for potential confounders. In addition, the LA stiffness of HFpEF patients with T2DM was higher than those without T2DM. After a median follow-up of 13.7 months, 46 patients (28.1%) reached the composite end point. LAS-peak (hazard ratios: 0.88; 95% confidence interval: 0.81–0.95; P = 0.001) was significantly associated with the risk of heart failure hospitalization or death after adjusting for demographic and clinical characteristics, LV global longitudinal strain, E/e’, and LA volume index. In contrast, other LA function and stiffness parameters did not independently predict the risk of adverse events. Kaplan-Meier analysis showed that HFpEF patients with T2DM and low LAS-peak (

Primary cardiac tumors (PCTs) are rare, with benign PCTs being relatively common in approximately 75% of all PCTs. Benign PCTs are usually asymptomatic, and they are found incidentally by imaging. Even if patients present with symptoms, they are usually nonspecific. Before the application of imaging modalities to the heart, our understanding of these tumors is limited to case reports and autopsy studies. The advent and improvement of various imaging technologies have enabled the non-invasive evaluation of benign PCTs. Although echocardiography is the most commonly used imaging examination, it is not the best method to describe the histological characteristics of tumors. At present, cardiac magnetic resonance (CMR) and cardiac computed tomography (CCT) are often used to assess benign PCTs providing detailed information on anatomical and tissue features. In fact, each imaging modality has its own advantages and disadvantages, multimodality imaging uses two or more imaging types to provide valuable complementary information. With the widespread use of multimodality imaging, these techniques play an indispensable role in the management of patients with benign PCTs by providing useful diagnostic and prognostic information to guide treatment. This article reviews the multimodality imaging characterizations of common benign PCTs.

Background: Right ventricular longitudinal strain of free wall (RV FWLS) assessed by two-dimensional speckle-tracking echocardiography (2D-STE) is recognized as an independent predictor of poor prognosis in patients with heart failure with preserved ejection fraction (HFpEF). However, the prognostic implications of three-dimensional STE (3D-STE) parameters in patients with HFpEF have not been well-established. The purpose of our study was to determine whether 3D-STE parameters were the more powerful predictors of poor outcomes in HFpEF patients compared with 2D-STE indices.Methods: Eighty-one consecutive patients with HFpEF were studied by 2D-STE and 3D-STE. RV volumes, ejection fraction (EF) and 3D-RVFWLS were measured by 3D-STE. 2D-RVFWLS was determined by 2D-STE. Patients were followed for the primary end point of heart failure (HF)-related hospitalization and death for HF.Results: After a median follow-up period of 17 months, 39 (48%) patients reached the end point of cardiovascular events. Compared with HFpEF patients without end-point events, those with end-point events had lower RVEF and 3D-RVFWLS (P < 0.05). Separate multivariate Cox regression analyses revealed that 3D-RVFWLS (HR 5.73; 95% CI 2.77–11.85; P < 0.001), RVEF (HR 3.47; 95% CI 1.47–8.21; P = 0.005), and 2D-RVFWLS (HR 3.17; 95% CI 1.54–6.53; P = 0.002) were independent predictors of adverse outcomes. The models with 3D-RVFWLS (AIC = 246, C-index = 0.75) and RVEF (AIC = 247, C-index = 0.76) had similar predictive performance for future clinical events as with 2D-RVFWLS (AIC = 248, C-index = 0.74).Conclusions: 3D-STE parameters are powerful predictors of poor outcomes, providing a similar predictive value as 2D-STE indices in patients with HFpEF. These findings support the potential of RV 3D-STE to identify HFpEF patients at higher risk for adverse cardiac events.

Atrial fibrillation (AF) is the most common arrhythmia with significant morbidity and mortality. Exacerbated by the aging population, the prevalence of AF is gradually increasing. Accurate evaluation of structure and function of left atrium (LA) has important prognostic significance in patients with AF. Echocardiography is the imaging technique of first choice to assess LA structure and function due to its better availability, accessibility and safety over cardiac computed tomography and cardiac magnetic resonance. Therefore, the aim of this review is to summarize the recent research progress of evaluating LA size by three-dimensional echocardiography and LA function by speckle tracking echocardiography (STE) in predicting the occurrence and recurrence of AF and determining the risk of stroke in AF. In addition, we summarized the role of traditional echocardiography in detecting AF patients that are at high risk of heart failure or cardiovascular death.

Patients with heart failure (HF) have high morbidity and mortality. Accurate assessment of right ventricular (RV) function has important prognostic significance in patients with HF. However, conventional echocardiographic parameters of RV function have limitations in RV assessments due to the complex geometry of right ventricle. In recent years, speckle tracking echocardiography (STE) has been developed as promising imaging technique to accurately evaluate RV function. RV longitudinal strain (RVLS) using STE, as a sensitive index for RV function evaluation, displays the powerfully prognostic value in patients with HF. Therefore, the aim of the present review was to summarize the utility of RVLS in patients with HF.

Integrated thin-film lithium niobate (LN) electro-optic (EO) modulators of broad bandwidth, low insertion loss, low cost and high production rate are essential elements in contemporary interconnection industries and disruptive applications. Here, we demonstrated the design and fabrication of a high performance thin-film LN EO modulator using photolithography assisted chemo-mechanical etching (PLACE) technology. Our device shows a 3-dB bandwidth over 50 GHz, along with a comparable low half wave voltage-length product of 2.16 Vcm and a fiber-to-fiber insertion loss of 2.6 dB. The PLACE technology supports large footprint, high fabrication uniformity, competitive production rate and extreme low device optical loss simultaneously, our result shows promising potential for developing high-performance large-scale low-loss photonic integrated devices.

The respiratory chain is very important for bacterial survival and pathogenicity, yet the roles of the respiratory chain in P. aeruginosa remain to be fully elucidated. Here, we not only proved experimentally that the operon PA4427-PA4431 of Pseudomonas aeruginosa PAO1 encodes respiratory chain complex III (cytobc1), but also found that it played important roles in virulence and pathogenicity. PA4429–31 deletion reduced the production of the virulence factors, including pyocyanin, rhamnolipids, elastase, and extracellular polysaccharides, and it resulted in a remarkable decrease in pathogenicity, as demonstrated in the cabbage and Drosophila melanogaster infection models. Furthermore, RNA-seq analysis showed that PA4429–31 deletion affected the expression levels of the genes related to quorum-sensing systems and the transport of iron ions, and the iron content was also reduced in the mutant strain. Taken together, we comprehensively illustrated the function of the operon PA4427–31 and its application potential as a treatment target in P. aeruginosa infection.

The large-scale cascade hydropower plants in southwestern China now challenge a multi-market environment in the new round of electricity market reform. They not only have to supply the load for the local provincial market, but also need to deliver electricity to the central and eastern load centers in external markets, which makes the generation scheduling much more complicated, with a correlated uncertain market environment. Considering the uncertainty of prices and correlation between multiple markets, this paper has proposed a novel optimization model of long-term generation scheduling for cascade hydropower plants in multiple markets to seek for the maximization of overall benefits. The Copula function is introduced to describe the correlation of stochastic prices between multiple markets. The price scenarios that obey the Copula fitting function are then generated and further reduced by using a scenario reduction strategy that combines hierarchical clustering and inconsistent values. The proposed model is applied to perform the long-term generation scheduling for the Wu River cascade hydropower plants and achieves an increase of 106.93 million yuan of annual income compared with the conventional scheduling model, without considering price scenarios, showing better performance in effectiveness and robustness in multiple markets.

Texture classification is an important topic for many applications in machine vision and image analysis, and Gabor filter is considered one of the most efficient tools for analyzing texture features at multiple orientations and scales. However, the parameter settings of each filter are crucial for obtaining accurate results, and they may not be adaptable to different kinds of texture features. Moreover, there is redundant information included in the process of texture feature extraction that contributes little to the classification. In this paper, a new texture classification technique is detailed. The approach is based on the integrated optimization of the parameters and features of Gabor filter, and obtaining satisfactory parameters and the best feature subset is viewed as a combinatorial optimization problem that can be solved by maximizing the objective function using hybrid ant lion optimizer (HALO). Experimental results, particularly fitness values, demonstrate that HALO is more effective than the other algorithms discussed in this paper, and the optimal parameters and features of Gabor filter are balanced between efficiency and accuracy. The method is feasible, reasonable, and can be utilized for practical applications of texture classification.

With the rapid development of the internet of things (IoT), traditional industries are setting off a massive wave of digitization. In the era of the Internet of Everything, millions of devices and links in IoT pose more significant challenges to data management. Most existing solutions employ centralized systems to control IoT devices, which brings about the privacy and security issues in IoT data management. Recently, blockchain has attracted much attention in the field of IoT due to its decentralization, traceability, and non-tamperability. However, it is non-trivial to apply the current blockchain techniques to IoT due to the lack of scalability and high resource costs. Different blockchain platforms have their particular advantages in the scenario of IoT data management. In this paper, we propose a cross-chain framework to integrate multiple blockchains for efficient and secure IoT data management. Our solution builds an interactive decentralized access model which employs a consortium blockchain as the control station. Other blockchain platforms customized for specific IoT scenarios run as the backbone of all IoT devices. It is equivalent to opening the off-chain channels on the consortium blockchain. Our model merges transactions in these channels for confirmation based on the notary mechanism. Finally, we implement a prototype of the proposed model based on hyperledge Fabric and IOTA Tangle. We evaluate the performance of our method through extensive experiments. The results demonstrate the effectiveness and efficiency of our framework.

Abstract Background Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. Objectives We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. Methods Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. Results PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. Conclusions These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease., Competing Interests: Declaration of interests K.M.N., M.L.L., and J.D.B. are listed as inventors on provisional US patent OKLA.P0023US.P1 filed on 09/13/23, which is related to the work described here., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immunogenic cell death (ICD) arises when cells are under stress, and their membranes are damaged. They release damage-associated molecular patterns (DAMPs) that stimulate and drive the type and magnitude of the immune response. In the presence of an antigen, DAMPs ride the longevity and efficacy of antigen-specific immunity. Yet, no tool can induce the controlled ICD with predictable results. A peptide-based tool, [II], is designed that aggregates in the cell and causes cell membrane damage, generates ICD and DAMPs release on various cell types, and hence can act as an adjuvant. An influenza vaccine is prepared by combining [II] with influenza hemagglutinin (HA) subunit antigens. The results show that [II] induced significantly higher HA-specific immunoglobulin G1 (IgG1) and IgG2a antibodies than HA-only immunized mice, while the peptide itself did not elicit antibodies. This paper demonstrates the first peptide-aggregation induced immunogenic rupture (PAIIR) approach as a vaccine adjuvant. PAIIR is a promising adjuvant with a high potential to promote universal protection upon influenza HA vaccination., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lang, Norman, Amadou Amani, Shadid, Ballard and Lang.)

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and, when conjugated to a protein carrier, induces a protective antibody response in animal models. Given that CD1d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18 -/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

The pathology associated with Clostridioides difficile disease is caused in large part by TcdB, an intracellular bacterial toxin that inactivates small GTPases. Despite C. difficile causing enteric disease, antitoxin IgG is a clear correlate of protection against infection-associated pathology. Immunization with TcdB-based immunogens or passive transfer of monoclonal antibodies specific for the TcdB carboxy-terminal domain (CTD) confers protection following C. difficile infection. Whether the mechanism by which circulating IgG is delivered to the gut depends on specific receptor-mediated transport or is solely reflective of infection-induced damage to the gut remains unclear. Here, we tested the hypothesis that neonatal Fc receptor (FcRn) is required for the delivery of systemic TcdB-specific IgG to the gut and protection against C. difficile-associated pathology. FcRn-expressing mice and FcRn-deficient littermates were immunized subcutaneously with Alhydrogel adjuvant-adsorbed CTD before challenge with live C. difficile spores. FcRn was required for the delivery of systemic TcdB-specific IgG to the gut and for vaccine-induced protection against C. difficile-associated disease. The lack of FcRn expression had minimal effects on the composition of the gut microbiome and did not affect susceptibility to C. difficile infection in nonimmunized mice. In further experiments, intraperitoneal injection of immune sera in FcRn-deficient mice led to the transport of protective IgG to the gut independently of infection, confirming a reported method of bypassing the FcRn. Our results reveal an FcRn-dependent mechanism by which systemic immunization-induced IgG protects the gut during enteric C. difficile infection. These findings may be beneficial for the targeting of C. difficile-specific IgG to the gut.

The CD1d-binding glycolipid α-galactosylceramide (α-GC) is a potent adjuvant that activates NKT cells and in turn enhances T-dependent humoral immunity. Very little is known about how NKT cells and the NKT follicular helper (NKTfh) subset influence the immune response to T-independent polysaccharides. In this study, we used a Cre-Lox approach to generate mice devoid of the Bcl6 master transcription factor in CD4 lineage cells and thus devoid of NKTfh cells but not total NKT cells. It was observed that α-GC-driven IgG1 class switch against a polysaccharide Ag was dependent on the NKTfh subset. However, α-GC was unable to stimulate a polysaccharide-specific Ab recall response. It was observed that NKT-derived IL-21 was able to exert limited influence on the IgG1 response and was therefore likely to work in concert with other factors. This work shows that α-GC-driven NKTfh cells can direct polysaccharide-specific B cell responses by promoting IgG1 class switch but do not provide signals needed for generation of polysaccharide-specific B cell memory., Competing Interests: DISCLOSURES The authors have no financial conflicts of interest.

Activation of iNKT cells with the CD1d-binding glycolipid adjuvant α-galactosylceramide (α-GC) enhances humoral immunity specific for coadministered T-dependent Ag. However, the relationship between the iNKT cell and the classic T helper (Th) or T follicular helper (Tfh) function following this immunization modality remains unclear. We show that immunization with the C-terminal domain (CTD) of Clostridium difficile toxin B (TcdB), accompanied by activation of iNKT cells with α-GC, led to enhanced production of CTD-specific IgG, which was CD1d- and iNKT cell-dependent and associated with increased neutralization of active TcdB. Immunization with CTD plus α-GC followed by NP hapten-linked CTD increased NP-specific IgG1 titers in an NKT-dependent manner, suggesting that iNKT activation could enhance Th or Tfh function or that iNKT and iNKTfh cells could provide supplemental, yet independent, B cell help. Th, Tfh, iNKT, and iNKTfh cells were, therefore, examined quantitatively, phenotypically, and functionally following immunization with CTD or with CTD plus α-GC. Our results demonstrated that α-GC-activated iNKT cells had no direct effect on the numbers, phenotype, or function of Th or Tfh cells. However, CD4 + T cell-specific ablation of the Bcl6 transcription factor demonstrated that Tfh and iNKTfh cells both contributed to B cell help. This work extends our understanding of the immune response to vaccination and demonstrates an important contribution by NKTfh cells to humoral immunity., (© Society for Leukocyte Biology.)

Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizing in vitro, but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cell memory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Alum-based adjuvants facilitate vaccine-driven humoral immunity, but their mechanism of action remains poorly understood. Herein, we report that lack of type II NKT cells is associated with intact, mature B cells but dampened humoral immunity following immunization with Alum-adsorbed T-dependent antigen. Type II NKT cells facilitated production of IL-4, IL-5, IL-10, IL-13, and antibody by LN and splenocyte cultures following Alum/antigen administration in vivo and antigen restimulation in vitro. Addition of IL-4 and IL-5 to type II NKT-deficient cultures restored in vitro antibody production. Intracellular staining revealed that Alum-primed type II NKT cells coordinated IL-4 secretion by T cells. Alum did not significantly affect CD1d expression in vivo, but addition of CD1d-blocking mAb diminished cytokine production and in vitro antibody production. Type II NKT cells therefore function as part of the Alum-sensing apparatus and in a CD1d-dependent manner, facilitate T(H)2-driven humoral immunity. This may have important consequences for understanding the mechanism of action of Alum-containing vaccines.

CD1d-restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T-dependent, antigen-derived peptide on MHC class II and CD1d-binding glycolipid α-GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d-expressing, DTR-transgenic DCs and CD1d(+) or CD1d(-) nontransgenic DCs. Following DT-mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d(+) and CD1d(-) DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d(+) DCs blocked in vivo expansion of antigen-specific cytotoxic (CD8(+)) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT-enhanced CD8(+) T cell expansion, it is dispensable for specific antibody production.

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4(-/-) mice and IFNγ(-/-) mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4(-/-) mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (alpha-galactosylceramide [alpha-GC]) at the time of immunization improves PA-specific Ab responses. We observed that alpha-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d(-/-) mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jalpha281(-/-) mice expressing CD1d but lacking type I alpha-GC-reactive NKT cells, alpha-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences.

Activation of natural killer-like T (NKT) cells with the CD1d ligand alpha-galactosylceramide enhances T-dependent humoral immune responses against coadministered T-dependent Ag. At present, there is little information on the mechanisms involved other than a dependence on CD1d expression by antigen-presenting cells and/or development of the NKT subset. We therefore tested the hypothesis that direct presentation of alpha-GC by B cells was required for NKT-enhanced Ab responses against T-dependent Ag. We reconstituted B cell-deficient microMT mice with B cells from C57Bl/6 donors or CD1d(-/-) donors before immunization with NP-KLH alone or NP-KLH mixed with alpha-GC. We made the surprising observation that B-cell expression of CD1d is absolutely required for the NKT-enhanced Ab response. Our data show that the mechanism by which NKT cells enhance humoral immune responses involves interaction with CD1d-expressing B cells.

Mutations in ras and p53 are the most prevalent mutations found in human nonmelanoma skin cancers. Although some p53 mutations cause a loss of function, most result in expression of altered forms of p53, which may exhibit gain-of-function properties. Therefore, understanding the consequences of acquiring p53 gain-of-function versus loss-of-function mutations is critical for the generation of effective therapies for tumors harboring p53 mutations. Here we describe an inducible mouse model in which skin tumor formation is initiated by activation of an endogenous K-ras(G12D) allele. Using this model we compared the consequences of activating the p53 gain-of-function mutation p53(R172H) and of deleting the p53 gene. Activation of the p53(R172H) allele resulted in increased skin tumor formation, accelerated tumor progression, and induction of metastasis compared with deletion of p53. Consistent with these observations, the p53(R172H) tumors exhibited aneuploidy associated with centrosome amplification, which may underlie the mechanism by which p53(R172H) exerts its oncogenic properties. These results clearly demonstrate that p53 gain-of-function mutations confer poorer prognosis than loss of p53 during skin carcinogenesis and have important implications for the future design of therapies for tumors that exhibit p53 gain-of-function mutations.

Specific interaction of class II/peptide with the T-cell receptor (TCR) expressed by class II-restricted CD4+ T helper (Th) cells is essential for in vivo production of antibodies reactive with T-dependent antigen. In response to stimulation with CD1d-binding glycolipid, Valpha14+ TCR-expressing, CD1d-restricted natural killer T (NKT) cells may provide additional help for antibody production. We tested the hypothesis that the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances production of antibodies reactive with T-dependent antigen in vivo. alpha-GC enhanced antibody production in vivo in a CD1d-dependent manner in the presence of class II-restricted Th cells and induced a limited antibody response in Th-deficient mice. alpha-GC also led to alterations in isotype switch, selectively increasing production of immunoglobulin G2b. Further analysis revealed that alpha-GC led to priming of class II-restricted Th cells in vivo. Additionally, we observed that alpha-GC enhanced production of antibodies reactive with T-independent antigen, showing the effects of NKT cells on B cells independently of Th cells. Our data show that NKT cells have multiple effects on the induction of a humoral immune response. We propose that NKT cells could be exploited for the development of novel vaccines where protective antibody is required.

CD1d is a non-polymorphic MHC class I-related protein that binds and presents glycolipid antigens to T cell antigen receptors expressed by NK-like T (NKT) cells. CD1d-dependent immune responses play critical roles in infectious disease, autoimmunity, allergy and cancer. We tested the hypothesis that B cell antigen receptor (BCR) targeting of a biotin-modified version of the CD1d-binding antigen alpha-galactosylceramide (biotin-alpha-GalCer) results in enhanced murine CD1d-mediated presentation as compared with presentation of non-targeted biotin-alpha-GalCer. Presentation of BCR-targeted antigen to NKT cells was enhanced 100- to 1000-fold compared with non-targeted antigen. CD1d presentation of BCR-targeted antigen was observed after 4 h treatment, consistent with a requirement for endosomal trafficking. Furthermore, unlike non-targeted antigen, BCR-targeted antigen was not loaded directly onto cell-surface CD1d. Blocking BCR signaling with the Syk tyrosine kinase inhibitor piceatannol inhibited presentation of BCR-targeted antigen but not non-targeted antigen. Piceatannol blocked transport of the BCR to CD1d-containing endosomes, showing that intersection of BCR-targeted antigen with endosomes is required for enhanced mCD1d antigen presentation. Our data suggest that the BCR facilitates capture of low quantities of mCD1d antigens to enhance CD1d-dependent immune responses.

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.

CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.

The human IgA Fc receptor (FcalphaR, CD89) triggers several important physiological functions, including phagocytosis, NADPH oxidase activation and antigen presentation. Efforts are underway to delineate FcalphaR signal-transduction pathways that control these functions. In a previous study, we demonstrated that cross-linking of FcalphaR increased its partitioning into membrane glycolipid rafts and was accompanied by gamma-chain-dependent recruitment and phosphorylation of the tyrosine kinases Lck/Yes-related novel protein tyrosine kinase (Lyn) and Bruton's tyrosine kinase (Btk). Here we have performed a more extensive characterization of signalling effectors recruited to rafts on FcalphaR cross-linking. We demonstrate that in addition to tyrosine kinases Lyn and Btk, FcalphaR cross-linking also recruits B-lymphocyte kinase (Blk) and spleen tyrosine kinase (Syk) to rafts. We show recruitment of phosphoinositide kinases, including 3-phosphoinositide 3-kinase and phospholipase Cgamma2, and serine/threonine kinases such as protein kinase C (PKC) alpha, PKCepsilon, and protein kinase B (PKB) alpha. This suggests that lipid rafts serve as sites for FcalphaR-triggered recruitment of multiple classes of signalling effectors. We further demonstrate that tyrosine kinases and PKCalpha have a sustained association with rafts, whereas phosphoinositide 3-kinase and its downstream effectors have a transient association with rafts. This is consistent with temporally regulated divergence of FcalphaR signalling pathways in rafts. Furthermore, we suggest the spatial separation of signalling effectors by transport of phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, PKBalpha and PKCepsilon to endocytic compartments containing internalized FcalphaR.

The Aspergillus nidulans NIMX(CDC2) protein kinase has been shown to be required for both the G(2)/M and G(1)/S transitions, and recent evidence has implicated a role for NIMX(CDC2) in septation and conidiation. While much is understood of its G(2)/M function, little is known about the functions of NIMX(CDC2) during G(1)/S, septation, and conidiophore development. In an attempt to better understand how NIMX(CDC2) is involved in these processes, we have isolated four extragenic suppressors of the A. nidulans nimX2(cdc2) temperature-sensitive mutation. Mutation of these suppressor genes, designated snxA-snxD for suppressor of nimX, affects nuclear division, septation, and conidiation. The cold-sensitive snxA1 mutation leads to arrest of nuclear division during G(1) or early S. snxB1 causes hyperseptation in the hyphae and sensitivity to hydroxyurea, while snxC1 causes septation in the conidiophore stalk and aberrant conidiophore structure. snxD1 leads to slight septation defects and hydroxyurea sensitivity. The additional phenotypes that result from the suppressor mutations provide genetic evidence that NIMX(CDC2) affects septation and conidiation in addition to nuclear division, and cloning and biochemical analysis of these will allow a better understanding of the role of NIMX(CDC2) in these processes.