Electrotaxis of alveolar epithelial cells in direct-current electric fields.

PURPOSE: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. METHODS: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. RESULTS: The successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. CONCLUSION: EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting.

PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION: AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Value of lung ultrasound score for evaluation of blast lung injury in goats.

PURPOSE: To establish a severe blast lung injury model of goats and investigate the feasibility of lung ultrasonic score in the evaluation of blast lung injury. METHODS: Twenty female healthy goats were randomly divided into three groups by different driving pressures: 4.0 MPa group (n = 4), 4.5 MPa group (n = 12) and 5.0 MPa group (n = 4). The severe blast lung injury model of goats was established using a BST-I bio-shock tube. Vital signs (respiration, heart rate and blood pressure), lung ultrasound score (LUS), PO2/FiO2 and extravascular lung water (EVLW) were measured before injury (0 h) and at 0.5 h, 3 h, 6 h, 9 h, 12 h after injury. Computed tomography scan was performed before injury (0 h) and at 12 h after injury for dynamic monitoring of blast lung injury and measurement of lung volume. The correlation of LUS with PaO2/FiO2, EVLW, and lung injury ratio (lesion volume/total lung volume*100%) was analyzed. All animals were sacrificed at 12 h after injury for gross observation of lung injury and histopathological examination. Statistical analysis was performed by the SPSS 22.0 software. The measurement data were expressed as mean ± standard deviation. The means of two samples were compared using independent-sample t-test. Pearson correlation analysis was conducted. RESULTS: (1) At 12 h after injury, the mortality of goats was 0, 41.67% and 100% in the 4.0 Mpa, 4.5 MPa and 5.0 MPa groups, respectively; the area of pulmonary hemorrhage was 20.00% ± 13.14% in the 4.0 Mpa group and 42.14% ± 15.33% in the 4.5 MPa group. A severe lung shock injury model was established under the driving pressure of 4.5 MPa. (2) The respiratory rate, heart rate, LUS and EVLW were significantly increased, while PaO2/FiO2 was significantly reduced immediately after injury, and then they gradually recovered and became stabilized at 3 h after injury. (3) LUS was positively correlated with EVLW (3 h: r = 0.597, 6 h: r = 0.698, 9 h: r = 0.729; p < 0.05) and lung injury ratio (12 h: r = 0.884, p < 0.05), negatively correlated with PaO2/FiO2 (3 h: r = -0.871, 6 h: r = -0.637, 9 h: r = -0.658; p < 0.05). CONCLUSION: We established a severe blast lung injury model of goats using the BST-I bio-shock tube under the driving pressure of 4.5 MPa and confirmed that ultrasound can be used for quick evaluation and dynamic monitoring of blast lung injury.

Physiological clearance of tau in the periphery and its therapeutic potential for tauopathies.

Accumulation of pathological tau is the hallmark of Alzheimer's disease and other tauopathies and is closely correlated with cognitive decline. Clearance of pathological tau from the brain is a major therapeutic strategy for tauopathies. The physiological capacity of the periphery to clear brain-derived tau and its therapeutic potential remain largely unknown. Here, we found that cisterna magna injected 131I-labelled synthetic tau dynamically effluxed from the brain and was mainly cleared from the kidney, blood, and liver in mice; we also found that plasma tau levels in inferior vena cava were lower than those in femoral artery in humans. These findings suggest that tau proteins can efflux out of the brain and be cleared in the periphery under physiological conditions. Next, we showed that lowering blood tau levels via peritoneal dialysis could reduce interstitial fluid (ISF) tau levels in the brain, and tau levels in the blood and ISF were dynamically correlated; furthermore, tau efflux from the brain was accelerated after the addition of another set of peripheral system in a parabiosis model. Finally, we established parabiosis mouse models using tau transgenic mice and their wild-type littermates and found that brain tau levels and related pathologies in parabiotic transgenic mice were significantly reduced after parabiosis, suggesting that chronic enhancement of peripheral tau clearance alleviates pathological tau accumulation and neurodegeneration in the brain. Our study provides the first evidence of physiological clearance of brain-derived pathological tau in the periphery, suggesting that enhancing peripheral tau clearance is a potential therapeutic strategy for tauopathies.

TNF-α release capacity is suppressed immediately after hemorrhage and resuscitation.

PURPOSE: It has been suggested that patients with traumatic insults are resuscitated into a state of an early systemic inflammatory response. We aimed to evaluate the influence of hemorrhagic shock and resuscitation (HSR) upon the inflammatory response capacity assessed by overall TNF-α secretion capacity of the host compared to its release from circulating leukocytes in peripheral circulation. METHODS: Rats (8/group) subjected to HS (MAP of 30-35 mmHg for 90 min followed by resuscitation over 50 min) were challenged with Lipopolysaccharide (LPS), 1 µg/kg intravenously at the end of resuscitation (HSR-LPS group) or 24 h later (HSR-LPS24 group). Control animals were injected with LPS without bleeding (LPS group). Plasma TNF-α was measured at 90 min after the LPS challenge. In addition, whole blood (WB) was obtained either from healthy controls (CON) immediately after resuscitation (HSR), or at 24 h post-shock (HSR 24). WB was incubated with LPS (100 ng/mL) for 2 h at 37 °C. TNF-α concentration and LPS binding capacity (LBC) was determined. RESULTS: Compared to LPS group, HSR followed by LPS challenge resulted in suppression of plasma TNF-α in HSR-LPS and HSR-LPS24 groups (1835 ± 478, 273 ± 77, 498 ± 200 pg/mL, respectively). Compared to CON the LPS-induced TNF-α release capacity of circulating leukocytes ex vivo was strongly declined both at the end of resuscitation (HSR) and 24 h later (HSR24) (1012 ± 259, 313 ± 154, 177 ± 63 ng TNF/mL, respectively). The LBC in WB was similar between CON and HSR and only moderately enhanced in HSR24 (57 ± 6, 56 ± 6, 71 ± 5 %, respectively). CONCLUSION: Our data suggest that the overall inflammatory response capacity is decreased immediately after HSR, persisting up to 24 h, and is independent of LBC.

Soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea attenuates bleomycin-induced pulmonary fibrosis in mice.

Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-ß1 (TGF-ß1), interleukin-1ß (IL-1ß) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-ß1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

Depletion of STYK1 inhibits intrahepatic cholangiocarcinoma development both in vitro and in vivo.

Intrahepatic cholangiocarcinoma (ICC) has been reported to be the second most common primary hepatic carcinoma worldwide, and very limited therapies are currently available. Serine threonine tyrosine kinase (STYK1), a member of the receptor tyrosine kinase family, exhibits tumorigenicity in many types of cancers and is a potential therapeutic target for ICC. In this study, STYK1 was knocked down in the ICC cell lines HCCC-9810 and RBE via a lentivirus-mediated system using short hairpin RNA (shRNA). Next, cell proliferation, colony formation, cell cycle progression, tumor formation in nude mice, migration and invasion, and the expression levels of cell cycle proteins in Lv-sh STYK1- or Lv-sh Con-infected cells were analyzed by CCK-8 assay, colony formation evaluation, flow cytometry, tumor formation evaluation, wound scratch assay, transwell assay, and western blotting. The results indicated that depletion of STYK1 inhibits ICC development both in vitro and in vivo.

Tumor suppressor Fbxw7 antagonizes WNT signaling by targeting ß-catenin for degradation in pancreatic cancer.

Pancreatic cancer is one of the deadliest solid malignancies associated with aberrant Wnt signaling activation. Fbxw7 mutations have been implicated in the development of pancreatic cancer, whereas the exact mechanism of this ubiquitin ligase as a tumor suppressor remains unclear in pancreatic carcinogenesis. Here, we describe that Fbxw7 is downregulated upon pancreatic cancer development. Depletion of Fbxw7 results in tumor suppression in pancreatic cancer cells, while Fbxw7 overexpression inhibits pancreatic cancer cell proliferation and invasion. Considering the negative correlation between Fbxw7 and ß-catenin, we find that Fbxw7 antagonizes Wnt signaling through targeting ß-catenin for its degradation. Moreover, the inhibitory effect of Fbxw7 on pancreatic cancer cell proliferation is mainly executed by the destruction of the Wnt/ß-catenin signaling pathway. We also reveal that c-myc, a widely accepted target of Fbxw7, is also transcriptionally regulated by the Fbxw7/ß-catenin axis in pancreatic cancer cells. Collectively, our results demonstrate that Fbxw7 is a novel regulator of Wnt/ß-catenin signaling-dependent regulation of pancreatic cancer cell growth and invasion, and inactivation of Fbxw7 in pancreatic cancer tissues might be the reason for the aberrant activation of Wnt signaling.

The cellular kinetics of lung alveolar epithelial cells and its relationship with lung tissue repair after acute lung injury.

BACKGROUND: Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. METHODS: Based on the fact that postnatal regeneration of alveolar tissue has been attributed to alveolar epithelial cells, we established a hemorrhagic shock and Lipopolysaccharide (LPS) lung injury model. Using this model, we analyzed the cellular kinetics of lung alveolar epithelial cells. RESULTS: The results showed that alveolar epithelium type 2 cells (AEC2s) are damage resistant during acute lung injury, they might be the main cells involved in lung injury and repair. Then we observed the relationship between the expression of HGF, c-Met following ALI in rat lung and proliferation of AEC2s. The proliferation of AEC2s was inhibited when isolated primary AEC2s were co-cultured with c-Met inhibitor SU11274. Furthermore, the numbers of AEC2s was significantly decreased when ALI rats were administrated with SU11274 in vivo. It provided further evidence that the HGF/c-Met signaling plays a vital role in ALI-induced AEC2s proliferation. CONCLUSIONS: AEC2s are damage resistant during acute lung injury and the HGF/c-Met signaling pathway is of vital importance in the proliferation of AEC2s after ALI.

Isolation of lung multipotent stem cells using a novel microfluidic magnetic activated cell sorting system.

In recent years, more and more research has shown that the lung is an organ of regenerative potential, with several types of stem/progenitor cells undergoing proliferation and differentiation after lung injury and participating the injury repair process. Mouse lung multipotent stem cells (MLSCs) have extensive self-renewal ability in culture and could differentiate into endothelial and lung epithelial (alveolar epithelial type 1, 2, and Clara) cells in vitro. But the research of MLSCs was limited due to its rarity. In this study, we introduced a novel microfluidic magnetic activated cell sorting system in the isolation of MLSCs. The sorted MLSCs had better viability and purity. They were identified by colony formation efficiency and differentiation ability and they have self-renewal and differentiation capacities, highlighting their stem cell properties.

Neutrophil CD64 expression as a diagnostic marker for sepsis in adult patients: a meta-analysis.

INTRODUCTION: Neutrophil CD64 (nCD64) expression appears to be a promising marker of bacterial infections. The aim of this meta-analysis was to assess the accuracy of nCD64 expression for the diagnosis of sepsis in critically ill adult patients. METHODS: We systematically searched PubMed, Embase, ISI Web of Knowledge, and the Cochrane Library for literature published between database inception and 19 May 2014, as well as reference lists of identified primary studies. Studies were included if they included assessment of the accuracy of nCD64 expression for sepsis diagnosis in adult patients and provided sufficient information to construct a 2×2 contingency table. RESULTS: A total of 8 studies comprising 1986 patients fulfilled the inclusion criteria for the final analysis. The pooled sensitivity and specificity were 0.76 (95 % confidence interval [CI], 0.73-0.78) and 0.85 (95 % CI, 0.82-0.87), respectively. The positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio were 8.15 (95 % CI, 3.82-17.36), 0.16 (95 % CI, 0.09-0.30), and 60.41 (95 % CI, 15.87-229.90), respectively. The area under the summary receiver operating characteristic curve of nCD64 expression with Q* value were 0.95 (Q* =0.89). CONCLUSIONS: On the basis of our meta-analysis, nCD64 expression is a helpful marker for early diagnosis of sepsis in critically ill patients. The results of the test should not be used alone to diagnose sepsis, but instead should be interpreted in combination with medical history, physical examination, and other test results.

Association between the TLR2 Arg753Gln polymorphism and the risk of sepsis: a meta-analysis.

INTRODUCTION: Recently, researchers in a number of studies have explored the association between the Toll-like receptor 2 (TLR2) Arg753Gln polymorphism and sepsis risk. However, the results were conflicting. In this meta-analysis, we aimed to confirm the effect of the TLR2 Arg753Gln polymorphism on sepsis risk. METHODS: Relevant records up to 1 June 2015 were retrieved from the PubMed, Embase, and Web of Knowledge databases. The odds ratios with their corresponding 95 % confidence intervals were used to assess the association between the TLR2 Arg753Gln polymorphism and sepsis risk. The selection of a fixed or random effects model was made according to a heterogeneity test in total and subgroup analyses. Sensitivity analysis and publication bias test were performed to ensure the reliability of our results. RESULTS: A total of 12 studies with aggregate totals of 898 cases and 1517 controls met our inclusion criteria for meta-analysis. There were significant associations between the TLR2 Arg753Gln polymorphism and sepsis risk in overall analyses under two genetic models (the allele comparison and the dominant model). In addition, subgroup analyses based on age group, ethnicity, sepsis type, and source of control also showed a significant effect of the TLR2 Arg753Gln polymorphism on sepsis risk. CONCLUSIONS: Our present meta-analysis supports a direct effect of the TLR2 Arg753Gln polymorphism on sepsis risk, especially in Europeans. The TLR2 Arg753Gln polymorphism might be used as a relevant risk estimate for the development of sepsis. Studies with larger sample sizes and homogeneous groups of patients with sepsis are required for further analysis.

Identification of haplotype tag single nucleotide polymorphisms within the nuclear factor-κB family genes and their clinical relevance in patients with major trauma.

INTRODUCTION: Nuclear factor-κB (NF-κB) family plays an important role in the development of sepsis in critically ill patients. Although several single nucleotide polymorphisms (SNPs) have been identified in the NF-κB family genes, only a few SNPs have been studied. METHODS: A total of 753 patients with major blunt trauma were included in this study. Tag SNPs (tSNPs) were selected from the NF-κB family genes (NFKB1, NFKB2, RELA, RELB and REL) through construction of haplotype blocks. The SNPs selected from genes within the canonical NF-κB pathway (including NFKB1, RELA and REL), which played a critical role in innate immune responses were genotyped using pyrosequencing method and analyzed in relation to the risk of development of sepsis and multiple organ dysfunction (MOD) syndrome. Moreover, the rs842647 polymorphism was analyzed in relation to tumor necrosis factor α (TNF-α) production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. RESULTS: Eight SNPs (rs28362491, rs3774932, rs4648068, rs7119750, rs4803789, rs12609547, rs1560725 and rs842647) were selected from the NF-κB family genes. All of them were shown to be high-frequency SNPs in this study cohort. Four SNPs (rs28362491, rs4648068, rs7119750 and rs842647) within the canonical NF-κB pathway were genotyped, and rs842647 was associated with sepsis morbidity rate and MOD scores. An association was also observed between the rs842647 A allele and lower TNF-α production. CONCLUSIONS: rs842647 polymorphism might be used as relevant risk estimate for the development of sepsis and MOD syndrome in patients with major trauma.

Rs1800625 in the receptor for advanced glycation end products gene predisposes to sepsis and multiple organ dysfunction syndrome in patients with major trauma.

INTRODUCTION: The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily, it plays pivotal roles in the pathogenesis of sepsis in several ways. Our previous study showed that rs1800625 (-429T/C) revealed a strong clinical relevance with sepsis morbidity rate and multiple organ dysfunction syndrome (MODS) in patients with major trauma. In this study, we enlarged the sample size, added two validation populations and examined the expression of RAGE on the surface of peripheral leukocytes to ex vivo lipopolysaccharide (LPS) stimulation in subjects with different genotypes. METHODS: Rs1800625 was genotyped using pyrosequencing in 837 Chinese Han patients with major trauma in Chongqing. We then validated the clinical relevance in 340 Zhejiang and 347 Yunnan patients. The expression of RAGE on the surface of peripheral blood mononuclear cells was measured by flow cytometric analysis. RESULTS: The results indicated that rs1800625 was significantly associated with sepsis morbidity rate and MODS in patients with major trauma in the Chongqing, Zhejiang and Yunnan districts. Patients with CC genotype had lower sepsis morbidity rate and MODS after major trauma. Furthermore, patients with CC genotype had significantly higher RAGE expression (P = 0.009). CONCLUSIONS: The rs1800625 polymorphism is a functional single nucleotide polymorphism and confers host susceptibility to sepsis and MODS in patients with major trauma.

Associations between interleukin-1 gene polymorphisms and sepsis risk: a meta-analysis.

BACKGROUND: Previous epidemiological studies have presented conflicting evidence regarding associations between interleukin-1 (IL-1) polymorphisms and sepsis susceptibility. We have performed a meta-analysis to evaluate possible associations between IL-1 polymorphisms and sepsis risk. METHODS: Eligible literature was retrieved from PubMed, Embase and Web of Knowledge databases until Jun 15, 2013. The pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using random-effects model in the overall and subgroup analysis based on ethnicity, sepsis severity and quality score. RESULTS: Eighteen studies addressing five IL-1 polymorphisms were included in this meta-analysis. For IL-1A-889 (rs1800587) polymorphism, significant association was observed in overall comparison for allelic effect (OR = 1.47, 95% CI = 1.01-2.13, P = 0.04). There were no significant associations between either IL-1B-511 (rs16944) or IL-1B-31 (rs1143627) and sepsis susceptibility in overall or subgroup analyses. For IL-1B + 3594 (rs143634) polymorphism, genotype TT decreased sepsis risk in overall analysis (OR = 0.59, 95% CI = 0.36-0.97, P = 0.04), as well as in Caucasian (OR = 0.57, 95% CI = 0.34-0.95, P = 0.03) and sepsis (OR = 0.55, 95% CI = 0.31-0.97, P = 0.04) subgroup analysis. For IL-1RN VNTR polymorphism, significant association was observed in overall comparison for allelic effect (OR = 1.40, 95% CI = 1.01-1.95, P = 0.04). Furthermore, the effect sizes of IL-1RN VNTR on sepsis risk increased with disease severity (septic shock OR > severe sepsis OR > sepsis OR). CONCLUSIONS: Our meta-analysis indicated that IL-1A-889, IL-1B + 3954 and IL-1RN VNTR might be associated with sepsis susceptibility. However, further studies with larger sample sizes and from homogenous populations would be necessary to validate these findings.

Role of ZIC1 methylation in hepatocellular carcinoma and its clinical significance.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in humans, and its prognosis is generally poor even after surgery. The zinc finger of the cerebellum (ZIC1) gene is a novel tumor suppressor gene that plays a crucial role in vertebrate development. Altered expression of ZIC1 is observed in various types of human cancers. The aims of the present study were to investigate the methylation status of ZIC1 in HCC and evaluate its clinical implication. The methylation status of ZIC1 was analyzed in 132 pairs of HCC and corresponding noncancerous tissues by methylation-specific polymerase chain reaction (PCR) (MSP). The expression of ZIC1 messenger RNA (mRNA) in HCC tissues was examined by real-time PCR. Methylation frequency of ZIC1 in HCC was significantly higher than that in the corresponding noncancerous tissues (P < 0.001), and it was correlated with tumor size (P = 0.022), histological differentiation (P = 0.033), and tumor stage (P = 0.009). The downregulation of the ZIC1 mRNA expression in HCC was correlated with the ZIC1 methylation (P < 0.001). The patients with methylated ZIC1 had a poorer overall survival than those without methylated ZIC1 (P < 0.001). Taken together, our results suggested that the hypermethylation may lead to promoter silencing of ZIC1 mRNA and associated with poor survival in HCC. Overall, aberrant methylation is an important mechanism for ZIC1 inactivation in HCC, and ZIC1 methylation may be a promising biomarker for the diagnosis and prognosis of HCC.

The correlation of expression levels of HIF-1α and HIF-2α in hepatocellular carcinoma with capsular invasion, portal vein tumor thrombi and patients' clinical outcome.

OBJECTIVES: The roles of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in the development of hepatocellular carcinoma have not been fully elucidated. Here, we aim to uncover the relationship between the prognosis of hepatocellular carcinoma patients and the expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in tumor tissues. METHODS: The protein levels of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α were detected by immunohistochemistry on paraffin sections of 126 paired hepatocellular carcinoma tissue and peritumoral tissue samples. The mRNA levels of them were detected by quantitative real-time polymerase chain reaction. RESULTS: High expression of hypoxia-inducible factor-1α was found in 57.1% (72/126) of tumor specimens, compared with 5.6% (7/126) in peritumoral tissues, while high expression of hypoxia-inducible factor-2α was found in only 13.5% (17/126) of tumors, compared with 47.6% (60/126) of peritumoral tissues. There was high expression of hypoxia-inducible factor-1α protein in hepatocellular carcinoma tissues closely associated with capsular infiltration and portal vein invasion, and thus lower overall survival and disease-free survival of hepatocellular carcinoma patients (P < 0.05). No significant association has been found between the expression of hypoxia-inducible factor-2α protein and capsular infiltration, portal vein invasion, overall survival and disease-free survival (P > 0.05). However, patients with high expression of both hypoxia-inducible factor-1α and hypoxia-inducible factor-2α have a significantly worse outcome than patients with low expression of both hypoxia-inducible factor-1α and hypoxia-inducible factor-2α (P < 0.05). CONCLUSIONS: The discordant results on expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression and stabilization mechanisms. The association between hypoxia-inducible factor-1α expression and unfavorable outcome indicates the importance of using hypoxia-inducible factor-1α as a treatment target in hepatocellular carcinoma.

Advancing skeletal health and disease research with single-cell RNA sequencing.

Orthopedic conditions have emerged as global health concerns, impacting approximately 1.7 billion individuals worldwide. However, the limited understanding of the underlying pathological processes at the cellular and molecular level has hindered the development of comprehensive treatment options for these disorders. The advent of single-cell RNA sequencing (scRNA-seq) technology has revolutionized biomedical research by enabling detailed examination of cellular and molecular diversity. Nevertheless, investigating mechanisms at the single-cell level in highly mineralized skeletal tissue poses technical challenges. In this comprehensive review, we present a streamlined approach to obtaining high-quality single cells from skeletal tissue and provide an overview of existing scRNA-seq technologies employed in skeletal studies along with practical bioinformatic analysis pipelines. By utilizing these methodologies, crucial insights into the developmental dynamics, maintenance of homeostasis, and pathological processes involved in spine, joint, bone, muscle, and tendon disorders have been uncovered. Specifically focusing on the joint diseases of degenerative disc disease, osteoarthritis, and rheumatoid arthritis using scRNA-seq has provided novel insights and a more nuanced comprehension. These findings have paved the way for discovering novel therapeutic targets that offer potential benefits to patients suffering from diverse skeletal disorders.

A functional variant of lipopolysaccharide binding protein predisposes to sepsis and organ dysfunction in patients with major trauma.

OBJECTIVE: To determine the hypothesis that genetic variations of the lipopolysaccharide-binding protein (LBP) gene influence risk for the development of sepsis and multiple organ dysfunction (MOD) in patients with major trauma. BACKGROUND: Lipopolysaccharide-binding protein plays a central role in innate immune response as the first line of defense and directing the microbial-induced activation of the inflammatory host response. Although a total of 112 single nucleotide polymorphisms (SNPs) have been identified so far within the entire LBP gene, only a few SNPs have been studied. METHODS: Nine haplotype tagging SNPs (htSNPs) were selected from 51 SNPs with a minor allele frequency of ≥5% using the HapMap database for the Chinese Han population. Two independent cohorts of major trauma patients were recruited. The 9 htSNPs were genotyped using pyrosequencing method and analyzed in relation to the risk of development of sepsis and MOD, LBP production, and lipopolysaccharide (LPS)-induced activation of peripheral blood leukocytes. Moreover, the functionality of the rs2232618 polymorphism was assessed by the observation of its effects on the binding and activation of LPS and the LBP-CD14 interaction. RESULTS: Among the 9 htSNPs, only the rs2232618 was significantly associated with higher susceptibility to sepsis and MOD in the 2 independent cohorts of major trauma patients recruited from southwest and eastern China. This SNP was also significantly associated with LPS-induced activation of peripheral blood leukocytes. In addition, the rs2232618 polymorphism could enhance LBP protein activities, showing significant increases in LPS binding to macrophages, LPS-induced cellular activation, and LBP-CD14 interaction at the presence of the variant LBP protein. CONCLUSIONS: The rs2232618 polymorphism is a functional SNP and confers host susceptibility to sepsis and multiple organ dysfunction in patients with major trauma.

Identification of haplotype tag single nucleotide polymorphisms within the receptor for advanced glycation end products gene and their clinical relevance in patients with major trauma.

INTRODUCTION: The receptor for advanced glycation end products (RAGE) has been considered as one of the major pattern recognition receptors and plays an important role in the development of sepsis and multiple organ dysfunction in critical illnesses. Although genetic variants of the RAGE gene have been shown to be well associated with susceptibility to some inflammatory diseases, little is known about their clinical relevance in the development of sepsis in critical ill patients. METHODS: Four genetic variants were selected from the entire RAGE gene and genotyped using pyrosequencing and polymerase chain reaction-length polymorphism methods. Association studies were performed in two independent Chinese Han populations. RESULTS: Among the four genetic variants, only the rs1800625 polymorphism was significantly associated with sepsis morbidity rate and multiple organ dysfunction (MOD) scores in patients with major trauma both in Chongqing (n = 496) and Zhejiang (n = 232) districts, respectively. Results from ex vivo responsiveness of peripheral blood leukocytes indicated that the rs1800625 polymorphism was well associated with decreased production of TNFα. In addition, the rs1800625 polymorphism could significantly inhibit the promoter activities of the RAGE gene. CONCLUSIONS: The rs1800625 polymorphism is a functional variant, which might be used as a relevant risk estimate for the development of sepsis and multiple organ dysfunction syndrome in patients with major trauma.