Specific Decrease in B-Cell-Derived Extracellular Vesicles Enhances Post-Chemotherapeutic CD8+ T Cell Responses.

Systemic immunosuppression greatly affects the chemotherapeutic antitumor effect. Here, we showed that CD19+ extracellular vesicles (EVs) from B cells through CD39 and CD73 vesicle-incorporated proteins hydrolyzed ATP from chemotherapy-treated tumor cells into adenosine, thus impairing CD8+ T cell responses. Serum CD19+ EVs were increased in tumor-bearing mice and patients. Patients with fewer serum CD19+ EVs had a better prognosis after chemotherapy. Upregulated hypoxia-inducible factor-1α (HIF-1α) promoted B cells to release CD19+ EVs by inducing Rab27a mRNA transcription. Rab27a or HIF-1α deficiency in B cells inhibited CD19+ EV production and improved the chemotherapeutic antitumor effect. Silencing of Rab27a in B cells by inactivated Epstein-Barr viruses carrying Rab27a siRNA greatly improved chemotherapeutic efficacy in humanized immunocompromised NOD PrkdcscidIl2rg-/- mice. Thus, decreasing CD19+ EVs holds high potential to improve the chemotherapeutic antitumor effect.

Erythrocytic α-Synuclein and the Gut Microbiome: Kindling of the Gut-Brain Axis in Parkinson's Disease.

BACKGROUND: Progressive spreading of α-synuclein via gut-brain axis has been hypothesized in the pathogenesis of Parkinson's disease (PD). However, the source of seeding-capable α-synuclein in the gastrointestinal tract (GIT) has not been fully investigated. Additionally, the mechanism by which the GIT microbiome contributes to PD pathogenesis remains to be characterized. OBJECTIVES: We aimed to investigate whether blood-derived α-synuclein might contribute to PD pathology via a gut-driven pathway and involve GIT microbiota. METHODS: The GIT expression of α-synuclein and the transmission of extracellular vesicles (EVs) derived from erythrocytes/red blood cells (RBCs), with their cargo α-synuclein, to the GIT were explored with various methods, including radioactive labeling of RBC-EVs and direct analysis of the transfer of α-synuclein protein. The potential role of microbiota on the EVs transmission was further investigated by administering butyrate, the short-chain fatty acids produced by gut microbiota and studying mice with different α-synuclein genotypes. RESULTS: This study demonstrated that RBC-EVs can effectively transport α-synuclein to the GIT in a region-dependent manner, along with variations closely associated with regional differences in the expression of gut-vascular barrier markers. The investigation further revealed that the infiltration of α-synuclein into the GIT was influenced significantly by butyrate and α-synuclein genotypes, which may also affect the GIT microbiome directly. CONCLUSION: By demonstrating the transportation of α-synuclein through RBC-EVs to the GIT, and its potential association with gut-vascular barrier markers and gut microbiome, this work highlights a potential mechanism by which RBC α-synuclein may impact PD initiation and/or progression. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Adhesion between EVs and tumor cells facilitated EV-encapsulated doxorubicin delivery via ICAM1.

Doxorubicin (Dox) is an anti-tumor drug with a broad spectrum, whereas the cardiotoxicity limits its further application. In clinical settings, liposome delivery vehicles are used to reduce Dox cardiotoxicity. Here, we substitute extracellular vesicles (EVs) for liposomes and deeply investigate the mechanism for EV-encapsulated Dox delivery. The results demonstrate that EVs dramatically increase import efficiency and anti-tumor effects of Dox in vitro and in vivo, and the efficiency increase benefits from its unique entry pattern. Dox-loading EVs repeat a "kiss-and-run" motion before EVs internalization. Once EVs touch the cell membrane, Dox disassociates from EVs and directly enters the cytoplasm, leading to higher and faster Dox import than single Dox. This unique entry pattern makes the adhesion between EVs and cell membrane rather than the total amount of EV internalization the key factor for regulating the Dox import. Furthermore, we recognize ICAM1 as the molecule mediating the adhesion between EVs and cell membranes. Interestingly, EV-encapsulated Dox can induce ICAM1 expression by irritating IFN-γ and TNF-α secretion in TME, thereby increasing tumor targeting of Dox-loading EVs. Altogether, EVs and EV-encapsulated Dox synergize via ICAM1, which collectively enhances the curative effects for tumor treatment.

Human γδ T cells induce CD8+ T cell antitumor responses via antigen-presenting effect through HSP90-MyD88-mediated activation of JNK.

Human Vγ9Vδ2 T cells have attracted considerable attention as novel alternative antigen-presenting cells (APCs) with the potential to replace dendritic cells in antitumor immunotherapy owing to their high proliferative capacity and low cost. However, the utility of γδ T cells as APCs to induce CD8+ T cell-mediated antitumor immune response, as well as the mechanism by which they perform APC functions, remains unexplored. In this study, we found that activated Vγ9Vδ2 T cells were capable of inducing robust CD8+ T cell responses in osteosarcoma cells. Activated γδ T cells also effectively suppressed osteosarcoma growth by priming CD8+ T cells in xenograft animal models. Mechanistically, we further revealed that activated γδ T cells exhibited increased HSP90 production, which fed back to upregulate MyD88, followed by JNK activation and a subsequent improvement in CCL5 secretion, leading to enhanced CD8+ T cell cross-priming. Thus, our study suggests that Vγ9Vδ2 T cells represent a promising alternative APC for the development of γδ T cell-based tumor immunotherapy.

Neddylation Alleviates Methicillin-Resistant Staphylococcus aureus Infection by Inducing Macrophage Reactive Oxygen Species Production.

Neddylation, a posttranslational modification in which NEDD8 is covalently attached to target proteins, has emerged as an endogenous regulator of innate immunity. However, the role of neddylation in methicillin-resistant Staphylococcus aureus (MRSA) infection remains unknown. In this study, we found that neddylation was activated after MRSA infection in vivo and in vitro. Inhibition of neddylation with MLN4924 promoted injury of liver and kidneys in C57BL/6 mice with MRSA bloodstream infection and increased mortality. Blockade of neddylation, either pharmacologically (MLN4924, DI591) or through the use of Uba3 small interfering RNA, inhibited Cullin3 neddylation and promoted Nrf2 accumulation, thus reducing reactive oxygen species (ROS) induction and bacterial killing ability in mouse peritoneal macrophages. In summary, our findings suggest that activation of neddylation in macrophages plays a critical protective role against MRSA infection by increasing ROS production, partially by signaling through the NEDD8-Cullin3-Nrf2-ROS axis. Furthermore, our results may provide a new non-antibiotic treatment strategy for MRSA infection through targeting of neddylation.

Optical image conversion and encryption based on structured light illumination and a diffractive neural network.

In this paper, an optical image encryption method is proposed based on structured light illumination and a diffractive neural network (DNN), which can realize conversion between different images. With the use of the structured phase mask (SPM) in the iterative phase retrieval algorithm, a plaintext image is encoded into a DNN composed of multiple phase-only masks (POMs) and ciphertext. It is worth noting that ciphertext is a visible image such that the conversion of one image to another is achieved, leading to high concealment of the proposed optical image encryption method. In addition, the wavelength of the illuminating light, all Fresnel diffraction distances, the optical parameters of the adopted SPM such as focal length and topological charge number, as well as all POMs in the DNN are all considered as security keys in the decryption process, contributing to a large key space and high level of security. Numerical simulations are performed to demonstrate the feasibility of the proposed method, and simulation results show that it exhibits high feasibility and safety as well as strong robustness.

α-Synuclein-containing erythrocytic extracellular vesicles: essential contributors to hyperactivation of monocytes in Parkinson's disease.

BACKGROUND: Immune system dysfunction, including higher levels of peripheral monocytes and inflammatory cytokines, is an important feature of Parkinson's disease (PD) pathogenesis, although the mechanism underlying the process remains to be investigated. In the central nervous system, it is well-known that α-synuclein (α-syn), a key protein involved in PD, activates microglia potently, and it is also reported that α-syn exists in the peripheral system, especially in erythrocytes or red blood cells (RBC) at exceedingly high concentration. The current study focused on the possibility that RBC-derived α-syn mediates the sensitization of peripheral monocytes in PD patients. METHODS: The hyperactivation of monocytes was assessed quantitatively by measuring mRNA levels of typical inflammatory cytokines (including IL-1ß, IL-6 and TNF-α) and protein levels of secreted inflammatory cytokines (including pro-inflammatory cytokines: IL-1ß, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13). Western blot, nanoparticle tracking analysis and electron microscopy were used to characterize RBC-derived extracellular vesicles (RBC-EVs). Inhibitors of endocytosis and leucine-rich repeat kinase 2 (LRRK2), another key protein involved in PD, were used to investigate how these two factors mediated the process of monocyte sensitization by RBC-EVs. RESULTS: Increased inflammatory sensitization of monocytes was observed in PD patients and PD model mice. We found that α-syn-containing RBC-EVs isolated from PD model mice or free form oligomeric α-syn induced the inflammatory sensitization of THP-1 cells, and demonstrated that endocytosis was a requirement for this pathophysiological pathway. Furthermore, the hyperactivation of THP-1 cells induced by RBC-EVs was associated with increased LRRK2 production and kinase activity. The phenomenon of inflammatory sensitization of human monocytes and increased LRRK2 were also observed by the treatment of RBC-EVs isolated from PD patients. CONCLUSIONS: Our data provided new insight into how hyperactivation of monocytes occurs in PD patients, and identified the central role played by α-syn-containing RBC-EVs in this process.

Security-enhanced multiple-image encryption based on quick response codes and modified double random phase encoding in the fractional Fourier transform domain.

A security-enhanced multiple-image encryption method is proposed based on quick response (QR) codes and modified double random phase encoding (DRPE) in the fractional Fourier transform (FrFT) domain in this paper, where each plaintext is first converted into QR code, and then each QR code is employed to generate the corresponding binary key for decryption with the help of random binary plaintext (RBP). Subsequently, the used RBP is encrypted into noise-like ciphertext by using the modified DRPE in the FrFT domain. In the modified DRPE method, the first random phase mask is activated by the initial FrFT with chaotic phase, and the wavelength of light and the fractional orders as well as the focal lengths of lenses are all used as digital keys to expand the key space. Moreover, the sensitivities of these digital keys are extremely high because the digital keys are closely mapped with the initial values of the chaotic system in the encryption process, which contributes to an extremely high security of the multiple-image encryption method. Furthermore, the high feasibility and strong robustness of the proposed security-enhanced multiple-image encryption method are also demonstrated by using computational simulations.

Glibenclamide Alleviates LPS-Induced Acute Lung Injury through NLRP3 Inflammasome Signaling Pathway.

Glibenclamide displays an anti-inflammatory response in various pulmonary diseases, but its exact role in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) remains unknown. Herein, we aimed to explore the effect of glibenclamide in vivo and in vitro on the development of LPS-induced ALI in a mouse model. LPS stimulation resulted in increases in lung injury score, wet/dry ratio, and capillary permeability in lungs, as well as in total protein concentration, inflammatory cells, and inflammatory cytokines including IL-1ß, IL-18 in bronchoalveolar lavage fluid (BALF), and lung tissues, whereas glibenclamide treatment reduced these changes. Meanwhile, the increased proteins of NLRP3 and Caspase-1/p20 after LPS instillation in lungs were downregulated by glibenclamide. Similarly, in vitro experiments also found that glibenclamide administration inhibited the LPS-induced upregulations in cytokine secretions of IL-1ß and IL-18, as well as in the expression of components in NLRP3 inflammasome in mouse peritoneal macrophages. Of note, glibenclamide had no effect on the secretion of TNF-α in vivo nor in vitro, implicating that its anti-inflammatory effect is relatively specific to NLRP3 inflammasome. In conclusion, glibenclamide alleviates the development of LPS-induced ALI in a mouse model via inhibiting the NLRP3/Caspase-1/IL-1ß signaling pathway, which might provide a new strategy for the treatment of LPS-induced ALI.

The deep learning model combining CT image and clinicopathological information for predicting ALK fusion status and response to ALK-TKI therapy in non-small cell lung cancer patients.

PURPOSE: This study aimed to investigate the deep learning model (DLM) combining computed tomography (CT) images and clinicopathological information for predicting anaplastic lymphoma kinase (ALK) fusion status in non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS: Preoperative CT images, clinicopathological information as well as the ALK fusion status from 937 patients in three hospitals were retrospectively collected to train and validate the DLM for the prediction of ALK fusion status in tumors. Another cohort of patients (n = 91) received ALK tyrosine kinase inhibitor (TKI) treatment was also included to evaluate the value of the DLM in predicting the clinical outcomes of the patients. RESULTS: The performances of the DLM trained only by CT images in the primary and validation cohorts were AUC = 0.8046 (95% CI 0.7715-0.8378) and AUC = 0.7754 (95% CI 0.7199-0.8310), respectively, while the DLM trained by both CT images and clinicopathological information exhibited better performance for the prediction of ALK fusion status (AUC = 0.8540, 95% CI 0.8257-0.8823 in the primary cohort, p < 0.001; AUC = 0.8481, 95% CI 0.8036-0.8926 in the validation cohort, p < 0.001). In addition, the deep learning scores of the DLMs showed significant differences between the wild-type and ALK infusion tumors. In the ALK-target therapy cohort (n = 91), the patients predicted as ALK-positive by the DLM showed better performance of progression-free survival than the patients predicted as ALK-negative (16.8 vs. 7.5 months, p = 0.010). CONCLUSION: Our findings showed that the DLM trained by both CT images and clinicopathological information could effectively predict the ALK fusion status and treatment responses of patients. For the small size of the ALK-target therapy cohort, larger data sets would be collected to further validate the performance of the model for predicting the response to ALK-TKI treatment.

Tumor-derived extracellular vesicles: Regulators of tumor microenvironment and the enlightenment in tumor therapy.

In recent decades, extracellular vesicles (EVs) have been proven to establish an important bridge of communication between cells or cells and their microenvironment. It is well known that EVs play crucial roles in many human diseases, especially in tumors. Tumor-derived EVs (TEVs) are not only involved in epithelial-mesenchymal transition and extracellular matrix remodeling to promote the invasion and metastasis, but also contribute to the suppression of antitumor immune responses by carrying different inhibitory molecules. In this review, we mainly discuss the effects of TEVs on the remodeling of tumor microenvironment through immune and non-immune associated mechanisms. We summarize the latest studies about utilizing EVs in clinical diagnosis and therapeutic drug delivery as well. In addition, the perspective of tumor therapy by targeting EVs is discussed in this review.

CD8α+CD11c+ Extracellular Vesicles in the Lungs Control Immune Homeostasis of the Respiratory Tract via TGF-ß1 and IL-10.

The immune balance of the respiratory tract is strictly regulated. Extracellular vesicles (EVs) have been reported to participate in maintaining the immune balance in the intestinal tract, but whether they are involved in regulation of the immune balance in the respiratory tract has yet to be revealed. In this study, we found that physiological EVs from lungs of WT mice (L-EVs) could be isolated, which contained the immunosuppressive cytokines TGF-ß1 and IL-10. Among L-EV subsets, only the CD8α+CD11c+ EV subset was positive for TGF-ß1 and IL-10 and could inhibit CD4+ T cell proliferation via TGF-ß1 in vitro and relieve murine asthmatic symptoms. Mechanistically, L-EVs were effective at inhibiting OVA peptide-specific CD4+ T cell proliferation in a TGF-ß1- and IL-10-dependent manner. In addition, they could prevent CD4+ T cells from hilar lymph nodes from secreting IL-4, IL-9, and IL-17A via IL-10 ex vivo, suggesting inhibition of Th2, Th9, and Th17 cell responses. Altogether, our results indicate that EVs from the lungs are involved in control of the immune balance in the respiratory tract, which reveals a novel mechanism in the maintenance of respiratory tract immune homeostasis.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Simulative study on speckle-spectral properties of a random pixelated grating.

Speckle spectrometers based on disordered media have shown great prospects and attracted more and more attention. To obtain a larger bandwidth and higher light-energy utilization ratio, a novel speckle spectrometer scheme is proposed based on a random pixelated grating. The speckle generation mechanism of the random pixelated grating is derived by using the diffraction and Fourier transform theory, and the speckle-spectral correlation properties are analyzed through numerical simulation. The influences of system parameters, such as variation ranges of grating periods and orientations, lens focal length, grating pixel size, and pixel number, are investigated. According to the simulation results, the speckle spectrometer system is optimized, and a spectral resolution of up to 0.01 nm can be achieved.

Dual-view holographic three-dimensional display using a single spatial light modulator and a directional light-guide plate composed of pixelated gratings.

In this paper, a dual-view holographic three-dimensional (3D) display using a single spatial light modulator (SLM) and a directional light-guide plate (DLGP) is proposed and implemented. The SLM is used to load the phase-only hologram calculated from two different 3D scenes for optical holographic reconstruction, and the DLGP composed of pixelated gratings with different periods and orientation angles is employed to guide the reconstructed images into two completely separated viewing zones, where different reconstructed perspectives in each viewing zone will form a stereoscopic 3D image. Furthermore, an experimental verification system for the proposed dual-view holographic 3D display is constructed, and the experimental results demonstrate that the proposed system can successfully present different 3D images in the left and right viewing zones simultaneously, verifying the feasibility of the proposed dual-view holographic 3D display.

Binocular holographic three-dimensional display using a single spatial light modulator and a grating.

In this paper, a binocular holographic three-dimensional (3D) display system combining a single spatial light modulator (SLM) and a grating is proposed and implemented. A synthetic phase-only hologram of the left and right 3D perspective images of an object is calculated by the layer-based Fresnel diffraction method according to the depth information, and uploaded onto the SLM for holographic 3D reconstruction with correct depth cues. The grating is designed and fabricated to guide the reconstructed left and right 3D perspective images to the corresponding eyes. Optical experiments demonstrate that the proposed system can successfully present binocular holographic 3D images with both the accommodation effect and binocular parallax, which enables observation free of the accommodation-vergence conflict and visual fatigue problem.

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection.

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis-dependent manner. Compared with autophagy-deficient Beclin-1(+/-) mice, we found that bone-marrow-derived DCs from wild-type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild-type BMDCs secreted higher levels of interleukin-6 (IL-6), tumour necrosis factor- α (TNF-α), interferon-ß (IFN-ß), IL-12p70 and IFN-γ than did Beclin-1(+/-) BMDCs. In contrast to Beclin-1(+/-) BMDCs, H1N1-infected WT BMDCs exhibited increased activation of extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and nuclear factor-κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co-localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL-6, TNF-α and IFN-ß in H1N1-infected BMDCs. In contrast to Beclin-1(+/-) BMDCs, H1N1-infected WT BMDCs were more efficient in inducing allogeneic CD4(+) T-cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4(+) Foxp3(+) regulatory T-cell differentiation. Moreover, WT BMDCs were more efficient at cross-presenting the ovalbumin antigen to CD8(+) T cells. We consistently found that Beclin-1(+/-) BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1-specific CD4(+) and CD8(+) T-cell responses, which produced lower levels of IL-6, TNF-α and IFN-ß in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.

Understanding the spatial and temporal patterns of copper in-use stocks in China.

Two approaches are adopted to characterize the comprehensive pattern of the copper in-use stocks in China. The top-down results indicate that both the total amount and the per capita quantity of the stocks have exhibited a significant and increasing trend for the past 60 years, especially since 2000. The top-down results show that the copper stocks increased from a negligible level of less than 1 kg/capita in 1952 to 44 kg/capita in 2012. The total stocks in 2012 are estimated to be 60 Mt by a top-down approach or 48 Mt by a bottom-up calculation. The bottom-up method determines that the largest reservoir is the infrastructure sector, which accounts for approximately 58% of the total stocks. The spatial pattern indicates that the copper in-use stocks are predominately spatially distributed in the eastern regions of China, a feature that is obviously different from the geographical distribution of the primary resources. Analysis on the prospects of stocks shows both the total magnitude and per capita value will continuously increase in the following decade, and enter a relatively stable stage in around 2030, with a maximum value of 106 kg/capita. The results improve the knowledge about closing copper cycles.

Exosomes with membrane-associated TGF-ß1 from gene-modified dendritic cells inhibit murine EAE independently of MHC restriction.

We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF-ß1 (sTGF-ß1-EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane-associated TGF-ß1 (mTGF-ß1-EXOs) and found mTGF-ß1-EXOs had more potent immunosuppressive activity than sTGF-ß1-EXOs in vitro. Treatment of mice with mTGF-ß1-EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE even after disease onset. Treatment of mice with mTGF-ß1-EXOs also impaired Ag-specific Th1 and IL-17 responses, but promoted IL-10 responses ex vivo. Treatment with mTGF-ß1-EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down-regulation of the p38, ERK, Stat3, and NF-κB activation and IL-6 expression in DCs. Treatment with mTGF-ß1-EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4(+)Foxp3(+)Treg cells from mTGF-ß1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-ß1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These results indicate that mTGF-ß1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.