The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

The R2R3-MYB transcription factor ZeMYB32 negatively regulates anthocyanin biosynthesis in Zinnia elegans.

KEY MESSAGE: This study identified an R2R3-MYB from Zinnia elegans, ZeMYB32, which negatively regulates anthocyanin biosynthesis.

The podoplanin-CLEC-2 interaction promotes platelet-mediated melanoma pulmonary metastasis.

BACKGROUND: Podoplanin (PDPN) expressed on tumour cells interacts with platelet C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate the role of the PDPN-platelet CLEC-2 interaction in melanoma pulmonary metastasis. METHODS: Murine melanoma B16-F0 cells, which have two populations that express podoplanin, were sorted by FACS with anti-podoplanin staining to obtain purified PDPN + and PDPN- B16-F0 cells. C57BL/6J mice transplanted with CLEC-2-deficient bone marrow cells were used for in vivo experiments. RESULTS: The in vivo data showed that the number of metastatic lung nodules in WT mice injected with PDPN + cells was significantly higher than that in WT mice injected with PDPN- cells and in WT or CLEC-2 KO mice injected with PDPN- cells. In addition, our results revealed that the platelet Syk-dependent signalling pathway contributed to platelet aggregation and melanoma metastasis. CONCLUSIONS: Our study indicates that the PDPN-CLEC-2 interaction promotes experimental pulmonary metastasis in a mouse melanoma model. Tumour cell-induced platelet aggregation mediated by the interaction between PDPN and CLEC-2 is a key factor in melanoma pulmonary metastasis.

Mechanotransduction activates canonical Wnt/ß-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves.

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/ß-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of ß-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/ß-catenin signaling in LECs. In turn, Wnt/ß-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking ß-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/ß-catenin signaling and, in turn, FOXC2.

Temperature regulation of carotenoid accumulation in the petals of sweet osmanthus via modulating expression of carotenoid biosynthesis and degradation genes.

BACKGROUND: Temperature is involved in the regulation of carotenoid accumulation in many plants. The floral color of sweet osmanthus (Osmanthus fragrans Lour.) which is mainly contributed by carotenoid content, is affected by temperature in autumn. However, the mechanism remains unknown. Here, to reveal how temperature regulates the floral color of sweet osmanthus, potted sweet osmanthus 'Jinqiu Gui' were treated by different temperatures (15 °C, 19 °C or 32 °C). The floral color, carotenoid content, and the expression level of carotenoid-related genes in petals of sweet osmanthus 'Jinqiu Gui' under different temperature treatments were investigated. RESULTS: Compared to the control (19 °C), high temperature (32 °C) changed the floral color from yellow to yellowish-white with higher lightness (L*) value and lower redness (a*) value, while low temperature (15 °C) turned the floral color from yellow to pale orange with decreased L* value and increased a* value. Total carotenoid content and the content of individual carotenoids (α-carotene, ß-carotene, α-cryptoxanthin, ß-cryptoxanthin, lutein and zeaxanthin) were inhibited by high temperature, but were enhanced by low temperature. Lower carotenoid accumulation under high temperature was probably attributed to transcriptional down-regulation of the biosynthesis gene OfPSY1, OfZ-ISO1 and OfLCYB1, and up-regulation of degradation genes OfNCED3, OfCCD1-1, OfCCD1-2, and OfCCD4-1. Up-regulation of OfLCYB1, and down-regulation of OfNCED3 and OfCCD4-1 were predicted to be involved in low-temperature-regulated carotenoid accumulation. Luciferase assays showed that the promoter activity of OfLCYB1 was activated by low temperature, and repressed by high temperature. However, the promoter activity of OfCCD4-1 was repressed by low temperature, and activated by high temperature. CONCLUSIONS: Our study revealed that high temperature suppressed the floral coloration by repressing the expression of carotenoid biosynthesis genes, and activating the expression of carotenoid degradation genes. However, the relative low temperature had opposite effects on floral coloration and carotenoid biosynthesis in sweet osmanthus. These results will help reveal the regulatory mechanism of temperature on carotenoid accumulation in the petals of sweet osmanthus.

A microscopic survey on microplastics in beverages: the case of beer, mineral water and tea.

It has been reported that microplastics exist ubiquitously in aquatic and terrestrial environments. Microplastic surveys on diverse daily foods with high consumption possibly containing microplastics have essential implications in clarifying the contamination routes, health risk assessment, and thereby preventing food pollution. Given the dependence of microplastic pollution on the regional environment, production and transportation, it further remains an open question on the number, size distribution and type of microplastics in foods from different countries worldwide. Here, we show that daily drinks produced worldwide, including beer, mineral water and tea, are all polluted with microplastics without exception. The number of microplastics investigated in this work lies in the range of 20-80 mL-1 for the beers, 10 mL-1 for the bottled mineral water, and 200-500 g-1 for the tea leaves. Quasi-spherical particles and irregular fragments dominate the shape of microplastics in beer and mineral water, whereas tea leaves carry numerous microplastic fibers. By identification through Raman spectroscopy, we observed the presence of polystyrene (PS) and polypropylene (PP) microplastics in beers, PP in bottled mineral water, and polyethylene (PE) and polyethylene terephthalate (PET) in tea leaves. Possible contamination sources include raw materials, atmosphere, and tools and containers that release microplastics. Given the facile adsorption of heavy metals and antibiotics to microplastics in beverages, public concern may arise regarding the accumulation of microplastics through the food chain and their synergetic harmful effect. Thus, our results should inspire further efforts that may contribute to the elimination and removal of microplastics from foods.

Genome-Wide Identification of Petunia HSF Genes and Potential Function of PhHSF19 in Benzenoid/Phenylpropanoid Biosynthesis.

Volatile benzenoids/phenylpropanoids are the main flower scent compounds in petunia (Petunia hybrida). Heat shock factors (HSFs), well known as the main regulator of heat stress response, have been found to be involved in the biosynthesis of benzenoid/phenylpropanoid and other secondary metabolites. In order to figure out the potential function of HSFs in the regulation of floral scent in petunia, we systematically identified the genome-wide petunia HSF genes and analyzed their expression and then the interaction between the key petunia HSF gene with target gene involved in benzenoid/phenylpropanoid biosynthesis. The results revealed that 34 HSF gene family members were obtained in petunia, and most petunia HSFs contained one intron. The phylogenetic analysis showed that 23 petunia HSFs were grouped into the largest subfamily HSFA, while only two petunia HSFs were in HSFC subfamily. The DBD domain and NLS motif were well conserved in most petunia HSFs. Most petunia HSF genes' promoters contained STRE motifs, the highest number of cis-acting element. PhHSF19 is highly expressed in petal tubes, followed by peduncles and petal limbs. During flower development, the expression level of PhHSF19 was dramatically higher at earlier flower opening stages than that at the bud stage, suggesting that PhHSF19 may have potential roles in regulating benzenoid/phenylpropanoid biosynthesis. The expression pattern of PhHSF19 is positively related with PhPAL2, which catalyzes the first committed step in the phenylpropanoid pathway. In addition, there are three STRE elements in the promoter of PhPAL2. PhHSF19 was proven to positively regulate the expression of PhPAL2 according to the yeast one hybrid and dual luciferase assays. These results lay a theoretical foundation for further studies of the regulation of HSFs on plant flower scent biosynthesis.

Slc35a1 deficiency causes thrombocytopenia due to impaired megakaryocytopoiesis and excessive platelet clearance in the liver.

Sialic acid is a common terminal residue of glycans on proteins and acidic sphingolipids such as gangliosides and has important biological functions. The sialylation process is controlled by more than 20 different sialyltransferases, many of which exhibit overlapping functions. Thus, it is difficult to determine the overall biological function of sialylation by targeted deletion of individual sialyltransferases. To address this issue, we established a mouse line with the Slc35a1 gene flanked by loxP sites. Slc35a1 encodes the cytidine-5'-monophosphate (CMP)-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation. Here we report our study regarding the role of sialylation on megakaryocytes and platelets using a mouse line with significantly reduced sialylation in megakaryocytes and platelets (Plt Slc35a1­ /­). The major phenotype of Plt Slc35a1­/­ mice was thrombocytopenia. The number of bone marrow megakaryocytes in Plt Slc35a1­/­ mice was reduced, and megakaryocyte maturation was also impaired. In addition, an increased number of desialylated platelets was cleared by Küpffer cells in the liver of Plt Slc35a1­/­ mice. This study provides new insights into the role of sialylation in platelet homeostasis and the mechanisms of thrombocytopenia in diseases associated with platelet desialylation, such as immune thrombocytopenia and a rare congenital disorder of glycosylation (CDG), SLC35A1-CDG, which is caused by SLC35A1 mutations.

Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans.

BACKGROUND: Sweet osmanthus (Osmanthus fragrans Lour.) is one of the top ten traditional ornamental flowers in China. The flowering time of once-flowering cultivars in O. fragrans is greatly affected by the relatively low temperature, but there are few reports on its molecular mechanism to date. A hypothesis had been raised that genes related with flower opening might be up-regulated in response to relatively low temperature in O. fragrans. Thus, our work was aimed to explore the underlying molecular mechanism of flower opening regulated by relatively low temperature in O. fragrans. RESULTS: The cell size of adaxial and abaxial petal epidermal cells and ultrastructural morphology of petal cells at different developmental stages were observed. The cell size of adaxial and abaxial petal epidermal cells increased gradually with the process of flower opening. Then the transcriptomic sequencing was employed to analyze the differentially expressed genes (DEGs) under different number of days' treatments with relatively low temperatures (19 °C) or 23 °C. Analysis of DEGs in Gene Ontology analysis showed that "metabolic process", "cellular process", "binding", "catalytic activity", "cell", "cell part", "membrane", "membrane part", "single-organism process", and "organelle" were highly enriched. In KEGG analysis, "metabolic pathways", "biosynthesis of secondary metabolites", "plant-pathogen interaction", "starch and sucrose metabolism", and "plant hormone signal transduction" were the top five pathways containing the greatest number of DEGs. The DEGs involved in cell wall metabolism, phytohormone signal transduction pathways, and eight kinds of transcription factors were analyzed in depth. CONCLUSIONS: Several unigenes involved in cell wall metabolism, phytohormone signal transduction pathway, and transcription factors with highly variable expression levels between different temperature treatments may be involved in petal cell expansion during flower opening process in response to the relatively low temperature. These results could improve our understanding of the molecular mechanism of relatively-low-temperature-regulated flower opening of O. fragrans, provide practical information for the prediction and regulation of flowering time in O. fragrans, and ultimately pave the way for genetic modification in O. fragrans.

Isolation and characterization of hexokinase genes PsHXK1 and PsHXK2 from tree peony (Paeonia suffruticosa Andrews).

Hexokinase (HXK) plays important roles in hexose phosphorylation and sugar signaling. HXK regulates the glucose-induced accumulation of anthocyanin in many species. Little is known about the biological function of the HXK gene family in Paeonia suffruticosa. cDNA sequences of two hexokinase genes PsHXK1 and PsHXK2 were isolated using RACE-PCR and RT-PCR from P. suffruticosa. PsHXK1 encodes 498 amino acids with a 1497-bp open reading frame (ORF), and PsHXK2 contains 493 amino acids with a 1482-bp ORF. Sequence and phylogenetic analyses suggest that PsHXK1 and PsHXK2 belong to type-B HXK and may function as glucose sensors. PsHXK1 and PsHXK2 mRNA were detected in all tested tissues. PsHXK1 is highly expressed in petals and stamens, while PsHXK2 is highly expressed in stamens. At the former stages of flower opening, PsHXK1 and PsHXK2 show higher expression levels in on-tree flowers compared with cut flowers. Overexpressing PsHXK1 and PsHXK2 in Arabidopsis enhances glucose sensitivity, inhibits plant growth in response to glucose, and induces anthocyanin accumulation in response to the high level of glucose. Overall, our results primarily reveal the biological function of PsHXK1 and PsHXK2, especially their involvement in glucose-induced anthocyanin accumulation.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Loss of mucin-type O-glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney.

The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1-derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1-derived O-glycans is catalyzed by the glycosyltransferase core 1 ß1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1-/-) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process-associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.

Plasma soluble podoplanin is a novel marker for the diagnosis of tumor occurrence and metastasis.

Podoplanin (PDPN) is expressed on many tumors and is involved in tumor metastasis. The objective of the present study was to develop an ELISA for determining soluble PDPN (sPDPN) levels as a potential novel tumor marker in plasma of patients with cancers for detection of tumor occurrence and metastasis. Mouse monoclonal antibodies (mAb) against human PDPN were developed and characterized. Two anti-PDPN mAb, SZ-163 and SZ-168, were used in a sandwich ELISA to detect plasma sPDPN in patients with cancers and in normal individuals. The levels of sPDPN were detected in patients with adenocarcinoma (87 cases, 31.09 ± 5.48 ng/ml), squamous cell carcinoma (86 cases, 6.91 ± 0.59 ng/ml), lung cancer (45 cases, 26.10 ± 7.62 ng/ml), gastric cancer (38 cases, 23.71 ± 6.90 ng/ml) and rectal cancer (27 cases, 32.98 ± 9.88 ng/ml), which were significantly higher than those in normal individuals (99 cases, 1.31 ± 0.13 ng/ml) (P < .0001). Moreover, the sPDPN levels in patients with metastatic cancers were higher (192 cases, 30.35 ± 3.63 ng/ml) than those in non-metastatic cancer patients (92 cases, 6.28 ± 0.77 ng/ml) (P < .0001). The post-treatment sPDPN levels of cancer patients (n = 156) (4.47 ± 0.35 ng/ml) were significantly lower compared with those seen pre-treatment (n = 128) (43.74 ± 4.97 ng/ml) (P < .0001). These results showed that an ELISA method was successfully established for quantitation of plasma sPDPN and plasma sPDPN levels correlate significantly with tumor occurrence and metastasis.

CLEC-2 and podoplanin, partners again.

In this issue of Blood, Tamura et al reveal a novel function for podoplanin on periarteriolar stromal cells in the bone marrow: promoting megakaryocyte growth and proplatelet formation by interaction with C-type lectin-like receptor 2 (CLEC-2).

O-glycans direct selectin ligands to lipid rafts on leukocytes.

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Intermediate-Temperature Creep Deformation and Microstructural Evolution of an Equiatomic FCC-Structured CoCrFeNiMn High-Entropy Alloy.

The tensile creep behavior of an equiatomic CoCrFeNiMn high-entropy alloy was systematically investigated over an intermediate temperature range (500-600 °C) and applied stress (140-400 MPa). The alloy exhibited a stress-dependent transition from a low-stress region (LSR-region I) to a high-stress region (HSR-region II). The LSR was characterized by a stress exponent of 5 to 6 and an average activation energy of 268 kJ mol-1, whereas the HSR showed much higher corresponding values of 8.9-14 and 380 kJ mol-1. Microstructural examinations on the deformed samples revealed remarkable dynamic recrystallization at higher stress levels. Dislocation jogging and tangling configurations were frequently observed in LSR and HSR at 550 and 600 °C, respectively. Moreover, dynamic precipitates identified as M23C6 or a Cr-rich σ phase were formed along grain boundaries in HSR. The diffusion-compensated strain rate versus modulus-compensated stress data analysis implied that the creep deformation in both stress regions was dominated by stress-assisted dislocation climb controlled by lattice diffusion. Nevertheless, the abnormally high stress exponents in HSR were ascribed to the coordinative contributions of dynamic recrystallization and dynamic precipitation. Simultaneously, the barriers imposed by these precipitates and severe initial deformation were referred to so as to increase the activation energy for creep deformation.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.