Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and ...prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca2+ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca2+-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specificTMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation byTMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specificTMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.
Recent developments in laboratory techniques adopting highly precise local instrumentation have made possible the determination of all five independent elastic parameters necessary for describing the ...small-strain stiffness in cross-anisotropic soils. However, the techniques and the derivation procedures are not necessarily straightforward, and different processes sometimes lead to apparently inconsistent sets of parameters, revealing their complex and sensitive nature. This paper firstly reports a new fully-instrumented triaxial system that was optimised to test Japanese standard-sized (∅70–75mm) clay samples. The testing techniques and procedures to obtain the five cross-anisotropic elastic parameters, defined in this paper for recoverable strain smaller than 0.001%, are reviewed. Some updates, including notes on how to deal with creep in soft clays and the optimisation of drained probe rates, are described. A simplified procedure is proposed for completing the parameter determination, without actually measuring the radial displacement, by inverting the relationship between the undrained Young׳s modulus and the drained elastic parameters in saturated soils. The validity of this approach is demonstrated by comparing the parameters of sedimentary clays obtained with and without radial measurements. By eliminating the need for complex radial instrumentation, this approach will make the quantification of stiffness anisotropy more accessible in less-equipped laboratories.
Distinct B cell populations, designated regulatory B (Breg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local ...inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified Breg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue Breg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support Breg cell function, and Breg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue Breg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that Breg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.
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•Regulatory B cells that constitutively produce IL-10 are present within adipose•Il10 deletion in B cells augments adipose tissue inflammation and insulin resistance•Adipose tissue environmental factors support the unique function of Breg cells•Breg cell function is compromised in obese adipose tissue
Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be ...simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones.
The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional ...platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4–5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b+ platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application.
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•Expandable megakaryocyte progenitors were established from human PSCs•BMI1 and BCL-XL suppress senescence and apoptosis induced by c-MYC in imMKCLs•imMKCLs differentiate into mature megakaryocytes and release functional platelets•Rapid and efficient platelet yield provides key advantages for clinical application
This study describes a clinically applicable strategy for the derivation of functional platelets from human induced pluripotent stem cells (iPSCs) using the establishment of stable immortalized megakaryocyte progenitor lines from the iPSCs.
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment ...strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is ...required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab′ fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.
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The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which ...transforming growth factor-1 (TGF-β1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-β1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6C
monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-β1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.
Towards the development of a mechanical model that can be part of multi-physical analysis of frozen soils, a program of systematic frozen-unfrozen parallel triaxial tests at different temperatures ...and strain rates was conducted. The mechanical behavior of the reconstituted high-plasticity clay samples was investigated and interpreted through a state concept based on Ladanyi and Morel’s (1990) postulate on the unique relationship between the inter-particle “effective” stress and the strain path. The Critical State Lines (CSLs) for clay specimens frozen undrained were mapped by referring to the shear behavior of unfrozen specimens sharing the same strain history. With other conditions set identical, the shear strength linearly increased with a decrease in the temperature for the range from −10°C to −2°C, and log-linearly increased with an increase in the strain rate for the range from 0.001%/min to 0.1%/min. Direct comparison of the strain-rate effects between frozen and unfrozen specimens with identical strain paths and states in the soil skeleton clearly indicates that the viscoplasticity derives from that of pore ice. A conceptual interpretative framework invoking temperature- and strain rate-dependent state bounding surfaces and CSLs was proposed to describe the behavior of frozen soils under steady and non-steady temperature and strain rate. The above observations of the behavioral features of frozen and unfrozen soils, with further experimental work, are expected to lead to the construction of a unified framework for describing the behavior under both states and the transition between them.
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