Single-cell atlas of the human neonatal small intestine affected by necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRß) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRß clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.

In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Exploring the dynamic adaptive responses of Epimedium pubescens to phosphorus deficiency by Integrated transcriptome and miRNA analysis.

Phosphorus, a crucial macronutrient essential for plant growth and development. Due to widespread phosphorus deficiency in soils, phosphorus deficiency stress has become one of the major abiotic stresses that plants encounter. Despite the evolution of adaptive mechanisms in plants to address phosphorus deficiency, the specific strategies employed by species such as Epimedium pubescens remain elusive. Therefore, this study observed the changes in the growth, physiological reponses, and active components accumulation in E. pubescensunder phosphorus deficiency treatment, and integrated transcriptome and miRNA analysis, so as to offer comprehensive insights into the adaptive mechanisms employed by E. pubescens in response to phosphorus deficiency across various stages of phosphorus treatment. Remarkably, our findings indicate that phosphorus deficiency induces root growth stimulation in E. pubescens, while concurrently inhibiting the growth of leaves, which are of medicinal value. Surprisingly, this stressful condition results in an augmented accumulation of active components in the leaves. During the early stages (30 days), leaves respond by upregulating genes associated with carbon metabolism, flavonoid biosynthesis, and hormone signaling. This adaptive response facilitates energy production, ROS scavenging, and morphological adjustments to cope with short-term phosphorus deficiency and sustain its growth. As time progresses (90 days), the expression of genes related to phosphorus cycling and recycling in leaves is upregulated, and transcriptional and post-transcriptional regulation (miRNA regulation and protein modification) is enhanced. Simultaneously, plant growth is further suppressed, and it gradually begins to discard and decompose leaves to resist the challenges of long-term phosphorus deficiency stress and sustain survival. In conclusion, our study deeply and comprehensively reveals adaptive strategies utilized by E. pubescens in response to phosphorus deficiency, demonstrating its resilience and thriving potential under stressful conditions. Furthermore, it provides valuable information on potential target genes for the cultivation of E. pubescens genotypes tolerant to low phosphorus.

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

CD4+ T-Cell Dysfunction in Severe COVID-19 Disease Is Tumor Necrosis Factor-α/Tumor Necrosis Factor Receptor 1-Dependent.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase.

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single-atom replacement in comparison to tyrosine. The arPTE-(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12-fold increase in kcat and 3.2-fold enhancement in kcat /KM at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

Prolonged Cold Ischemia Induces Necroptotic Cell Death in Ischemia-Reperfusion Injury and Contributes to Primary Graft Dysfunction after Lung Transplantation.

Primary graft dysfunction (PGD) is a major cause of morbidity and mortality after lung transplantation. Ischemia-reperfusion injury (IRI) is a key event that contributes to PGD, though complex interactions affect donor lungs status, such as preceding brain death (BD), hemorrhagic shock (HS), and pre-engraftment lung management, the latter recognized as important risk factors for PGD. We hypothesized that a multi-hit isogenic mouse model of lung transplantation is more closely linked to PGD than IRI alone. Left lung transplants were performed between inbred C57BL/6 mice. A one-hit model of IRI was established by inducing cold ischemia (CI) of the donor lungs at 0°C for 1, 72, or 96 hours before engraftment. Multi-hit models were established by inducing 24 hours of HS and/or 3 hours of BD before 24 hours of CI. The recipients were killed at 24 hours after transplant and lung graft samples were analyzed. In the one-hit model of IRI, up to 72-hour CI time resulted in minimal cellular infiltration near small arteries after 24-hour reperfusion. Extension of CI time to 96 hours led to increased cellular infiltration and necroptotic pathway activation, without evidence of apoptosis, after 24-hour reperfusion. In a multi-hit model of PGD, "HS + BD + IRI" demonstrated increased lung injury, cellular infiltration, and activation of necroptotic and apoptotic pathways compared with IRI alone. Treatment with an inhibitor of receptor-interacting protein kinase 1 kinase, necrostatin-1, resulted in a significant decrease of downstream necroptotic pathway activation in both single- and multi-hit models of IRI. Thus, activation of necroptosis is a central event in IRI after prolonged CI, though it may not be sufficient to cause PGD alone. Pathological evaluation of donor lungs after CI-induced IRI, in conjunction with pre-engraftment donor lung factors in our multi-hit model, demonstrated early evidence of lung injury consistent with PGD. Our findings support the premise that pre-existing donor lung status is more important than CI time alone for inflammatory pathway activation in PGD, which may have important clinical implications for donor lung retrieval.

Overexpression of Forkhead box Q1 correlates with poor prognosis in papillary thyroid carcinoma.

OBJECTIVE: Forkhead box Q1 (FOXQ1), a member of the forkhead transcription factor family, plays important parts in cell cycle, apoptosis, metabolism, immunology and tumour genesis. Its expression has been associated with poor clinical prognosis in various tumours. However, the clinical significance of FOXQ1 in papillary thyroid carcinoma (PTC) has not been fully studied. The purpose of this study was to investigate whether FOXQ1 is correlated with poor prognosis in PTC. DESIGN/METHODS: We performed a retrospective study of 136 PTCs. Immunohistochemistry (IHC) was used to examine the expression of FOXQ1 in 136 PTCs and 47 nodular goitre specimens. Rank-sum test, chi-square test, Kaplan-Meier survival analysis, univariate and multivariate Cox analyses were used to investigate the clinical and prognostic significance of FOXQ1 expression in PTC. RESULTS: The comparison of PTC specimens with nodular goitre with papillary hyperplasia specimens revealed an upregulation of FOXQ1 in PTC. Overexpression of FOXQ1 was observed in 63.24% of PTC and correlated with classic variant, tall variant, distant metastasis, AJCC stage and recurrence. FOXQ1-positive expression was associated with shorter disease-free survival: median disease-free survival of FOXQ1-positive patients was 23 months compared with 128 months for FOXQ1-negative patients (Log-rank χ2  = 12.31, P = 0.00045). Additional independent risk factors in this study were multifocality (recurrence-free survival [RFS]: hazard ratio [HR] = 2.391, P < 0.05), extrathyroidal extension (RFS: HR = 3.906, P < 0.05) and positive expression of FOXQ1 (RFS: HR = 6.385, P < 0.01). CONCLUSIONS: Our results indicated that FOXQ1 may be a useful additional biomarker to evaluate the progression of PTC and to predict likely relapse of disease.

Epigenetic Regulation of IL-17-Induced Chemokines in Lung Epithelial Cells.

Epithelial cells are known to have barrier functions in multiple organs and regulate innate immune responses. Airway epithelial cells respond to IL-17 by altering their transcriptional profiles and producing antimicrobial proteins and neutrophil chemoattractants. Although IL-17 has been shown to promote inflammation through stabilizing mRNA of CXCR2 ligands, how IL-17 exerts its downstream effects on its target cells through epigenetic mechanisms is largely unknown. Using primary human bronchial epithelial cells and immortalized epithelial cell line from both human and mouse, we demonstrated that IL-17-induced CXCR2 ligand production is dependent on histone acetylation specifically through repressing HDAC5. Furthermore, the chemokine production induced by IL-17 is strictly dependent on the bromodomain and extraterminal domain (BET) family as BET inhibition abolished the IL-17A-induced proinflammatory chemokine production, indicating a pivotal role of the recognition of acetylated histones. In combination with single-cell RNA-seq analysis, we revealed that the cell lines we employed represent specific lineages and their IL-17 responses were regulated differently by the DNA methylation mechanisms. Taken together, our data strongly support that IL-17 sustains epithelial CXCR2 ligand production through epigenetic regulation and the therapeutic potential of interrupting histone modification as well as the recognition of modified histones could be evaluated in neutrophilic lung diseases.

Tanshinone IIA therapeutically reduces LPS-induced acute lung injury by inhibiting inflammation and apoptosis in mice.

AIM: To study the effects of tanshinone IIA (TIIA) on lipopolysaccharide (LPS)-induced acute lung injury in mice and the underlying mechanisms. METHODS: Mice were injected with LPS (10 mg/kg, i.p.), then treated with TIIA (10 mg/kg, i.p.). Seven hours after LPS injection, the lungs were collected for histological study. Protein, LDH, TNF-α and IL-1ß levels in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in lungs were measured. Cell apoptosis and Bcl-2, caspase-3, NF-κB and HIF-1α expression in lungs were assayed. RESULTS: LPS caused marked histological changes in lungs, accompanied by significantly increased lung W/D ratio, protein content and LDH level in BALF, and Evans blue leakage. LPS markedly increased neutrophil infiltration in lungs and inflammatory cytokines in BALF. Furthermore, LPS induced cell apoptosis in lungs, as evidenced by increased TUNEL-positive cells, decreased Bcl-2 content and increased cleaved caspase-3 content. Moreover, LPS significantly increased the expression of NF-κB and HIF-1α in lungs. Treatment of LPS-injected mice with TIIA significantly alleviated these pathological changes in lungs. CONCLUSION: TIIA alleviates LPS-induced acute lung injury in mice by suppressing inflammatory responses and apoptosis, which is mediated via inhibition of the NF-κB and HIF-1α pathways.

[Protective effect of formula of removing both phlegm and blood stasis on myocardial tissues of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome].

OBJECTIVE: To study the protective effect of formula of removing both phlegm and blood stasis (TYTZ) on myocardial tissues of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. METHOD: Totally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Danlou tablet group, and TYTZ groups with doses of 2.0, 1.0, 0.5 g x kg(-1), with six in each group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary heart disease model of phlegm-stasis cementation syndrome in Chinese mini-swine. After the operation, they were administered with drugs for 8 weeks. The SOD activity and MDA content of each group were observed at the 0th week (before the experiment), the 2nd week after the high-fat diet (before the operation or drug administration) , the 6th week after the high-fat diet (4 weeks after the drug administration) and the 10th week after the high-fat diet (8 weeks after the drug administration). Meanwhile, the myocardial enzymogram test and the HE staining pathological observation were performed at the end of the experiment. The changes in the myocardial cell ultra-structure were observed under transmission electron microscope. RESULT: Compared with the normal control group, the model group showed significant decrease in serum SOD activity and notable increase in MDA content from the 2nd week to the end of experiment (P < 0.05 and P < 0.01). In the 10th week, the CK, LDH and CK-MB levels in serum also significantly increased in the model group (P < 0.05 and P < 0.01), with obvious structural abnormality in myocardial tissue pathologic morphology and ultra-structure. Compared with the model group, TYTZ groups showed specific increase in serum SOD activity and oblivious decrease in the MDA level (P < 0.05 or P < 0.01). Meanwhile, TYTZ could significantly decrease serum CK and LDH levels in the model group (P < 0.05 or P < 0.01), attenuate the ischemia injury of myocardial tissue, and improve the ultra-structure of cardiomyocytes. CONCLUSION: TYTZ shows an obvious protective effect on the myocardial injury in Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. Its mechanism is related to the resistance against free radical oxidation injury and the inhibition of the lipid per-oxidation.

Factor H preserves alternative complement function during ARDS, linked to improved survival.

Background: Effective regulation of complement activation may be crucial to preserving complement function during acute respiratory distress syndrome (ARDS). Factor H is the primary negative regulator of the alternative pathway of complement. We hypothesised that preserved factor H levels are associated with decreased complement activation and reduced mortality during ARDS. Methods: Total alternative pathway function was measured by serum haemolytic assay (AH50) using available samples from the ARDSnet Lisofylline and Respiratory Management of Acute Lung Injury (LARMA) trial (n=218). Factor B and factor H levels were quantified using ELISA using samples from the ARDSnet LARMA and Statins for Acutely Injured Lungs from Sepsis (SAILS) (n=224) trials. Meta-analyses included previously quantified AH50, factor B and factor H values from an observational registry (Acute Lung Injury Registry and Biospecimen Repository (ALIR)). Complement C3, and complement activation products C3a and Ba plasma levels were measured in SAILS. Results: AH50 greater than the median was associated with reduced mortality in meta-analysis of LARMA and ALIR (hazard ratio (HR) 0.66, 95% CI 0.45-0.96). In contrast, patients in the lowest AH50 quartile demonstrated relative deficiency of both factor B and factor H. Relative deficiency of factor B (HR 1.99, 95% CI 1.44-2.75) or factor H (HR 1.52, 95% CI 1.09-2.11) was associated with increased mortality in meta-analysis of LARMA, SAILS and ALIR. Relative factor H deficiency was associated with increased factor consumption, as evidenced by lower factor B and C3 levels and Ba:B and C3a:C3 ratios. Higher factor H levels associated with lower inflammatory markers. Conclusions: Relative factor H deficiency, higher Ba:B and C3a:C3 ratios and lower factor B and C3 levels suggest a subset of ARDS with complement factor exhaustion, impaired alternative pathway function, and increased mortality, that may be amenable to therapeutic targeting.

Qing-Xin-Jie-Yu Granule inhibits ferroptosis and stabilizes atherosclerotic plaques by regulating the GPX4/xCT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qing-Xin-Jie-Yu Granule (QXJYG) is an integrated traditional Chinese medicine formula used to treat atherosclerotic (AS) cardiovascular diseases. A randomized controlled trial found that QXJYG reduced cardiovascular events and experiments also verified that QXJYG attenuated AS by remodeling the intestinal flora. AIM OF THE STUDY: To determine whether QXJYG would attenuate AS and plaque vulnerability by regulating ferroptosis in high-fat diet-induced atherosclerotic ApoE-/- mice and to investigate the effects of QXJYG on macrophage ferroptosis in RAS-selective lethal 3 (RSL3)-induced J744A.1 cells. METHODS: AS models in ApoE-/- mice and RSL3-induced ferroptosis in J744A.1 cells were established to measure the protective and anti-ferroptotic effects of QXJYG in vivo and in vitro. The glutathione peroxidase 4 (GPX4)/cystine glutamate reverse transporter (xCT) signal pathway was examined by immunohistochemistry and western blotting. RESULTS: QXJYG attenuated AS progression and plaque vulnerability. Characteristic morphological changes of ferroptosis in the QXJYG-treated animals were rare. Total iron was significantly lower in the QXJYG group than in the model group (P < 0.05); QXJYG suppressed the lipid peroxidation (LPO) levels (malondialdehyde), enhanced the antioxidant capacity (superoxide dismutase and glutathione), and reduced inflammatory factors (interleukin [IL]-6, IL-1ß, tumor necrosis factor-α) associated with ferroptosis. Expression of GPX4/xCT in aorta tissues was remarkably increased in the QXJYG group. QXJYG inhibited ferroptosis in J744A.1 macrophages disturbed using RSL3. The Fe2+, LPO, and reactive oxygen species levels were lower in the QXJYG group than in the RSL3 group (P < 0.05). The QXJYG group showed higher expression of the GPX4/xCT signal pathway. CONCLUSION: QXJYG inhibits ferroptosis in vulnerable AS plaques partially via the GPX4/xCT signaling pathway.

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

BOA: A partitioned view of genome assembly.

De novo genome assembly is a fundamental problem in computational molecular biology that aims to reconstruct an unknown genome sequence from a set of short DNA sequences (or reads) obtained from the genome. The relative ordering of the reads along the target genome is not known a priori, which is one of the main contributors to the increased complexity of the assembly process. In this article, with the dual objective of improving assembly quality and exposing a high degree of parallelism, we present a partitioning-based approach. Our framework, BOA (bucket-order-assemble), uses a bucketing alongside graph- and hypergraph-based partitioning techniques to produce a partial ordering of the reads. This partial ordering enables us to divide the read set into disjoint blocks that can be independently assembled in parallel using any state-of-the-art serial assembler of choice. Experimental results show that BOA improves both the overall assembly quality and performance.

Involvement of Gax gene in hypoxia-induced pulmonary hypertension, proliferation, and apoptosis of arterial smooth muscle cells.

Hypoxia down-regulates the expression of the growth arrest-specific homeobox (Gax) in pulmonary arterial smooth muscle cells (PASMCs), resulting in increased cell proliferation and decreased apoptosis, but the mechanism for this response remains unclear. The present study investigated the role of Gax in the development of hypoxia-induced pulmonary hypertension (PH). We found that hypoxia suppressed the expression of endogenous Gax in rats, but not in those pretreated intratracheally with a Gax construct (Ad-Gax). Hypoxic rats pretreated with Ad-Gax were resistant to hypoxia-induced PH, right ventricular hypertrophy, increased wall thickness, and the muscularization of pulmonary arterioles. Hypoxia-induced PASMC proliferation and suppression of Gax expression were blocked by the Mitogen-activated protein kinase (MEK) inhibitor U0126. The PASMCs with Ad-Gax transfection exhibited hyperexpression of the Bcl-2-associated X protein (Bax) and hypoexpression of B-cell lymphoma 2 (Bcl-2), leading to cell apoptosis. Thus, our data indicate that the enhanced expression of Gax inhibits hypoxia-induced PASMC proliferation, probably via the extracellular-signal-regulated kinase (ERK) 1/2 pathway, and induces the apoptosis of hypoxic PASMCs via the Bcl-2/Bax pathway. Gax may be a potential new therapeutic target for pulmonary hypertension.

Deletion of peroxiredoxin 6 potentiates lipopolysaccharide-induced acute lung injury in mice.

OBJECTIVE: To investigate the role and signaling pathway of peroxiredoxin 6, a newly identified peroxidase, in lipopolysaccharide-induced acute lung injury. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory. SUBJECTS: Peroxiredoxin 6 (-/-) and wild-type C57BL/6 mice. INTERVENTIONS: Wild-type or peroxiredoxin 6 (-/-) mice were challenged by intratracheal instillation of lipopolysaccharide (5 mg/kg) for 4 hrs or 24 hrs for lung injury measurement. In other studies, peritoneal macrophages, isolated from wild-type and peroxiredoxin 6 (-/-) mice, were preincubated in presence or absence of mitogen-activated protein kinases inhibitors for 30 mins before being stimulated with lipopolysaccharide (1 µg/mL) for 4 hrs. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage myeloperoxidase activity and the lung injury score were significantly increased in peroxiredoxin 6 (-/-) mice compared with wild-type mice after lipopolysaccharide instillation at both 4 hrs and 24 hrs. Hydrogen peroxide and malondialdehyde levels, as well as nuclear factor-κB activities, tumor necrosis factor-α, interleukin-1ß, and matrix metalloproteinase-9 messenger RNA, protein concentration, and activities were significantly increased whereas total antioxidative capability was markedly decreased in lungs of peroxiredoxin 6 (-/-) mice compared with wild-type mice. In vitro studies showed intracellular reactive oxygen species levels and release of tumor necrosis factor-α, interleukin-1, and matrix metalloproteinase-9 were significantly increased in macrophages from peroxiredoxin 6 (-/-) mice compared with that from wild-type mice after lipopolysaccharide stimulation. Cytokines release was partially suppressed by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors, but not by the p38 mitogen-activated protein kinase inhibitor. CONCLUSIONS: Deletion of peroxiredoxin 6 exaggerates lipopolysaccharide-induced acute lung injury and inflammation with increased oxidative stress, inflammatory responses, and matrix degradation, all of which were partially dependent on nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun N-terminal kinase pathways.

[3.0 T magnetic resonance imaging of pancreatic cancer in an orthotopic nude murine model].

OBJECTIVE: To assess the methods of establishing an orthotopic nude murine model of human pancreatic cancer and explore the manifestations of MRI (magnetic resonance imaging) and its pathological fundamentals so as to provide research rationales for human pancreatic cancer. METHODS: The BXPC-3 cell of human pancreatic cancer was orthotopically planted in nude mice. And the animals were examined by a clinical 3.0 T magnetic resonance imager. The MRI examinations were analyzed along with their pathological findings. RESULTS: Among 18 mice, 15 of them grew tumors with a success ratio of 83.3%. The pathological findings conformed to those of high differentiation pancreatic parenchyma cancer. Comparing with the neighbor muscles, the tumors were homogeneous (66.7%, 10/15) or heterogeneous (33.3%, 5/15) of hypointense on T1-weighted images while homogeneous (26.7%, 4/15) or heterogeneous (73.3%, 11/15) hyperintense on T2-weighted images with heterogeneous enhancement. The border became obviously enhanced and there was mild central enhancement while the necrotic part showed no enhancement. CONCLUSION: 3.0T MRI can detect pancreatic neoplasms ≥ 2 mm and visualize clearly their locations, shapes, dimensions and internal structures in an orthotopic nude murine model. Thus it provides a visible framework for further studies of human pancreatic cancer.