An Intramolecular Rotor-Bridged Dimeric Cyanine Photothermal Transducer for Efficient Near-Infrared II Fluorescence Imaging-Guided Mitochondria-Targeted Phototherapy.

Near-infrared (NIR) heptamethine cyanine (HCy) dyes are promising photothermal transducers for image-guided cancer treatment owing to their prominent photophysical properties and high photothermal conversion ability. However, HCy photothermal transducers usually have poor photostability due to degradation induced by the self-generated reactive oxygen species. Herein, a novel mitochondria-targeting dimeric HCy dye, named dimeric oBHCy, is rationally designed, exhibiting strong near-infrared II (NIR-II) fluorescence emission, high photothermal conversion efficiency (PCE), and excellent photostability. The large π-conjugation and drastic intramolecular motion of the diphenol rotor in the dimeric oBHCy enhance the nonradiative energy dissipation and suppress the intersystem crossing process, thereby achieving a high PCE (49.2%) and improved photostability. Impressively, dimeric oBHCy can precisely target mitochondria and induce mitochondrial damage upon NIR light irradiation. Under the guidance of in vivo NIR-II fluorescence imaging, efficient NIR light-activated photothermal therapy of 4T1 breast tumors is accomplished with a tumor inhibitory rate of 96% following a single injection of the dimeric oBHCy. This work offers an innovative strategy for designing cyanine photothermal transducers with integrated NIR-II fluorescence and photothermal properties for efficient cancer theranostics.

An Active Self-Mitochondria-Targeting Cyanine Immunomodulator for Near-Infrared II Fluorescence Imaging-Guided Synergistic Photodynamic Immunotherapy.

Photodynamic therapy targeting mitochondria represents a promising therapeutic strategy for fighting diverse types of cancers. However, the currently available photosensitizers (PSs) suffer from insufficient therapeutic potency, limited mitochondria delivery efficiency, and the inability to treat invisible metastatic distal cancers. Herein, an active self-mitochondria-targeting heptapeptide cyanine (HCy) immunomodulator (I2HCy-QAP) is reported for near-infrared II (NIR-II) fluorescence imaging-guided photodynamic immunotherapy of primary and distal metastatic cancers. The I2HCy-QAP is designed by introducing a quaternary ammonium salt with a phenethylamine skeleton (QAP) into the iodinated HCy photosensitizer. The I2HCy-QAP can precisely target mitochondria due to the lipophilic cationic QAP unit, present strong NIR-II fluorescence tail emission, and effectively generate singlet oxygen 1O2 under NIR laser irradiation, thereby inducing mitochondria-targeted damages and eliciting strong systemic immunogenic cell death immune responses. The combination of the I2HCy-QAP-mediated photodynamic immunotherapy with anti-programmed death-1 antibody therapy achieves remarkable therapeutic efficacy against both primary and distal metastatic cancers with significant inhibition of lung metastasis in a triple-negative breast cancer model. This work provides a new concept for designing high-performance NIR emissive cyanine immunomodulators for NIR-II fluorescence-guided photodynamic immunotherapy.

Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins.

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

Characterization and Expression Analysis of Four Cadmium-Tolerance-Associated Genes of Avicennia marina (Forsk.).

Mangroves colonize the intertidal area of estuaries (e.g., Pichavaram, Payardia, and Mai Po) with remarkable cadmium (Cd) pollution. A study on the mechanism of mangrove plant response to Cd pollution can help to understand the adaptive characteristics of plants under Cd stress. This study explored the roles of peroxidase (PRX), pectate lyase (PL), and phytosulfokine (PSK) genes in cadmium tolerance of mangrove Avicennia marina. Full-length sequences of four genes (i.e., AmPRX1, AmPRX2, AmPL, and AmPSK) associated with metal tolerance were identified with suppression subtractive hybridization and rapid amplification of cDNA ends. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins. Real-time quantitative PCR analysis demonstrated that cadmium exposure resulted in differences in expression patterns among the tissues. Our findings emphasize the complex regulatory mechanism of these four genes in response to trace metal pollution and reveal their functions in metabolic signaling during the stress response.

Antibody-Drug Conjugates Targeting the Human Epidermal Growth Factor Receptor Family in Cancers.

Members of the human epidermal growth factor receptor (HER) family, which includes HER1 (also known as EGFR), HER2, HER3 and HER4, have played a central role in regulating cell proliferation, survival, differentiation and migration. The overexpression of the HER family has been recognized as one of the most common cellular dysregulation associated with a wide variety of tumor types. Antibody-drug conjugates (ADCs) represent a new and promising class of anticancer therapeutics that combine the cancer specificity of antibodies with cytotoxicity of chemotherapeutic drugs. Two HER2-directed ADCs, trastuzumane-emtansine (T-DM1) and trastuzumab-deruxtecan (DS-8201a), have been approved for HER2-positive metastatic breast cancer by the U.S. Food and Drug Administration (FDA) in 2013 and 2019, respectively. A third HER2-directed ADC, disitamab vedotin (RC48), has been approved for locally advanced or metastatic gastric or gastroesophageal junction cancer by the NMPA (National Medical Products Administration) of China in 2021. A total of 11 ADCs that target HER family receptors (EGFR, HER2 or HER3) are currently under clinical trials. In this review article, we summarize the three approved ADCs (T-DM1, DS-8201a and RC48), together with the investigational EGFR-directed ADCs (ABT-414, MRG003 and M1231), HER2-directed ADCs (SYD985, ARX-788, A166, MRG002, ALT-P7, GQ1001 and SBT6050) and HER3-directed ADC (U3-1402). Lastly, we discuss the major challenges associated with the development of ADCs, and highlight the possible future directions to tackle these challenges.

Comparative efficacy and safety of transurethral laser surgery with holmium laser, KTP laser, 2-micron laser or thulium laser for the treatment of non-muscle invasive bladder carcinoma: a protocol of network meta-analysis.

INTRODUCTION: The potential of transurethral laser surgery in treating non-muscle invasive bladder cancer (NMIBC) has been confirmed, however which types of lasers may be preferentially prescribed remains a debate. The aim of this network meta-analysis is to investigate the comparative efficacy and safety of transurethral laser surgery with four common types of laser including holmium laser, potassium titanylphosphate (KTP) laser, 2-micron laser or thulium laser for the treatment of NMIBC. METHODS AND ANALYSIS: A systematic search will be conducted to search all potentially eligible randomised controlled trials comparing different transurethral laser surgeries with each other or with standard transurethral resection among patients with NMIBC in PubMed, Embase, the Cochrane library, China National Knowledge Infrastructure, Wanfang database and Chongqing VIP from their inception until 31 May 2021. Two reviewers will be asked to independently select eligible studies, and assess the risk of bias of individual study with Cochrane risk of bias assessment tool. A random-effects network meta-analysis based on Markov chain Monte Carlo method will be carried out. Ranking probabilities will be considered to rank all laser types. Quantitative analysis will be carried out by using WinBUGS V.1.4.3. ETHICS AND DISSEMINATION: Ethical approval is not required because this is a network meta-analysis of published data. We will submit all findings to some conferences for preliminary communication and to a peer-reviewed journal for publication. TRIAL REGISTRATION NUMBER: 10.17605/OSF.IO/TD9MW.

The Wheat Wall-Associated Receptor-Like Kinase TaWAK-6D Mediates Broad Resistance to Two Fungal Pathogens Fusarium pseudograminearum and Rhizoctonia cerealis.

The soil-borne fungi Fusarium pseudograminearum and Rhizoctonia cerealis are the major pathogens for the economically important diseases Fusarium crown rot (FCR) and sharp eyespot of common wheat (Triticum aestivum), respectively. However, there has been no report on the broad resistance of wheat genes against both F. pseudograminearum and R. cerealis. In the current study, we identified TaWAK-6D, a wall-associated kinase (WAK) which is an encoding gene located on chromosome 6D, and demonstrated its broad resistance role in the wheat responses to both F. pseudograminearum and R. cerealis infection. TaWAK-6D transcript induction by F. pseudograminearum and R. cerealis was related to the resistance degree of wheat and the gene expression was significantly induced by exogenous pectin treatment. Silencing of TaWAK-6D compromised wheat resistance to F. pseudograminearum and R. cerealis, and repressed the expression of a serial of wheat defense-related genes. Ectopic expression of TaWAK-6D in Nicotiana benthamiana positively modulated the expression of several defense-related genes. TaWAK-6D protein was determined to localize to the plasma membrane in wheat and N. benthamiana. Collectively, the TaWAK-6D at the plasma membrane mediated the broad resistance responses to both F. pseudograminearum and R. cerealis in wheat at the seedling stage. This study, therefore, concludes that TaWAK-6D is a promising gene for improving wheat broad resistance to FCR and sharp eyespot.

The Receptor-like Kinase TaCRK-7A Inhibits Fusarium pseudograminearum Growth and Mediates Resistance to Fusarium Crown Rot in Wheat.

The fungus F. pseudograminearum can cause the destructive disease Fusarium crown rot (FCR) of wheat, an important staple crop. Functional roles of FCR resistance genes in wheat are largely unknown. In the current research, we characterized the antifungal activity and positive-regulatory function of the cysteine-rich repeat receptor-like kinase TaCRK-7A in the defense against F. pseudograminearum in wheat. Antifungal assays showed that the purified TaCRK-7A protein inhibited the growth of F. pseudograminearum. TaCRK-7A transcript abundance was elevated after F. pseudograminearum attack and was positively related to FCR-resistance levels of wheat cultivars. Intriguingly, knocking down of TaCRK-7A transcript increased susceptibility of wheat to FCR and decreased transcript levels of defense-marker genes in wheat. Furthermore, the transcript abundances of TaCRK-7A and its modulated-defense genes were responsive to exogenous jasmonate treatment. Taken together, these results suggest that TaCRK-7A can directly inhibit F. pseudograminearum growth and mediates FCR-resistance by promoting the expression of wheat defense genes in the jasmonate pathway. Thus, TaCRK-7A is a potential gene resource in FCR-resistant wheat breeding program.

Gastroprotective effect of cirsilineol against hydrochloric acid/ethanol-induced gastric ulcer in rats.

This study was designed to evaluate the gastroprotective activity of cirsilineol in hydrochloric acid (HCl)/ethanol-induced gastric ulcer model. Cirsilineol was administered at the doses of 20 and 40 mg/kg in HCl/ethanol-induced rats. The gastroprotective ability was verified by determining the ulcer score, total acidity, hemoglobin, inflammatory cytokines, lipid peroxides, and enzymatic antioxidants superoxide dismutase (SOD) and catalase (CAT) in gastric tissue and serum biochemical analysis. The results showed a favorable increase in the hemoglobin level, antioxidant enzymes (SOD and CAT), restored electrochemical balance (carbon dioxide & anion gap) while a noticeable decrease in ulcer index, total acidity, lipid peroxides, inflammatory cytokines (interleukin-1 beta [IL-1ß], IL-6, and tumor necrosis factor alpha) in rats treated with the cirsilineol. The serum biochemical analysis on liver markers (alkaline phosphatases, alanine aminotransferase, and aspartate aminotransferase), kidney markers (urea, creatinine, albumin, globulin, total protein), and lipid profile (triglyceride, high-density lipoprotein, total cholesterol) were attenuated by cirsilineol treatment in rats. Histopathology showed enhanced gastric protection and preserved the integrity of gastric mucosa upon cirsilineol administration. These results ultimately suggest that cirsilineol has gastroprotective effects that prevent the development of gastric ulcer.

Extracellular Vesicle-Transported Long Non-Coding RNA (LncRNA) X Inactive-Specific Transcript (XIST) in Serum is a Potential Novel Biomarker for Colorectal Cancer Diagnosis.

BACKGROUND Colorectal cancer (CRC) cell-derived extracellular vesicles (EVs) contribute to tumor progression. Differentially expressed long non-coding (lnc)RNAs may serve as biomarkers for CRC diagnosis. This study aimed to discuss the diagnostic value of serum EV-derived lncRNA X inactive-specific transcript (XIST) in CRC. MATERIAL AND METHODS Serum EVs were extracted and identified. Microarray analysis was performed to screen out the differentially expressed lncRNAs in serum EVs. The expression and diagnostic efficacy of the most differentially expressed lncRNA were measured. Kaplan-Meier survival analysis was performed to evaluate the association between survival time and XIST expression in EVs. The expression profile of serum EV-carried XIST in 94 CRC patients with different tumor-node-metastasis stages, lymph node metastasis, and differentiation was assessed. The serum contents of CEA, CA242, CA199, and CA153 were measured. RESULTS XIST in serum EVs in CRC patients was upregulated, with greatest diagnostic value. CRC patients with higher expression of XIST in serum EVs had worse 5-year survival rates and shorter life cycles, lower differentiation, higher lymph node metastasis, and tumor-node-metastasis than patients with lower XIST expression. XIST expression in serum EVs was positively correlated with CRC marker contents. CONCLUSIONS XIST upregulation in serum EVs is related to CRC progression, which may be helpful to the clinical diagnosis and prognosis of CRC.

The Cysteine-Rich Repeat Protein TaCRR1 Participates in Defense against Both Rhizoctonia cerealis and Bipolaris sorokiniana in Wheat.

The domain of unknown function 26 (DUF26), harboring a conserved cysteine-rich motif (C-X8-C-X2-C), is unique to land plants. Several cysteine-rich repeat proteins (CRRs), belonging to DUF26-containing proteins, have been implicated in the defense against fungal pathogens in ginkgo, cotton, and maize. However, little is known about the functional roles of CRRs in the important staple crop wheat (Triticum aestivum). In this study, we identified a wheat CRR-encoding gene TaCRR1 through transcriptomic analysis, and dissected the defense role of TaCRR1 against the soil-borne fungi Rhizoctonia cerealis and Bipolaris sorokiniana, causal pathogens of destructive wheat diseases. TaCRR1 transcription was up-regulated in wheat towards B. Sorokiniana or R. cerealis infection. The deduced TaCRR1 protein contained a signal peptide and two DUF26 domains. Heterologously-expressed TaCRR1 protein markedly inhibited the mycelia growth of B. sorokiniana and R. cerealis. Furthermore, the silencing of TaCRR1 both impaired host resistance to B. sorokiniana and R. cerealis and repressed the expression of several pathogenesis-related genes in wheat. These results suggest that the TaCRR1 positively participated in wheat defense against both B. sorokiniana and R. cerealis through its antifungal activity and modulating expression of pathogenesis-related genes. Thus, TaCRR1 is a candidate gene for improving wheat resistance to B. sorokiniana and R. cerealis.

The Eno Gene of Burkholderia cenocepacia Strain 71-2 is Involved in Phosphate Solubilization.

Bacterial strain 71-2 with phosphate-solubilizing activity was isolated from tobacco rhizosphere and classified as Burkholderia cenocepacia based on sequence analyses of 16S rRNA and recA genes. To learn phosphate-solubilizing mechanisms of 71-2, mutants showing reduced solubilizing phosphate activity were obtained using the EZ-Tn5 transposon. Mutant 71-2-MT51 was reduced in the solubilizing phosphate content to 34.36% as compared with the wild-type strain 71-2. The disrupted gene in 71-2-MT51 was cloned and sequenced, and the putative protein from the gene shared 65.26% identity to protein sequences of enolase from Escherichia coli, which suggests the gene encodes an enzyme of enolase. Complementation analyzing showed that Eno was responsible for phosphate solubilizing for B. cenocepacia strain 71-2. To our knowledge, this is the first report of Eno involved in phosphate solubilizing in B. cenocepacia as well as in other bacteria.

Root activities and arsenic translocation of Avicennia marina (Forsk.) Vierh seedlings influenced by sulfur and iron amendments.

Sulfur and iron are abundant and have close, complex interactions with the biogeochemical cycle of arsenic (As) in mangrove ecosystems. A hydroponic experiment was conducted to investigate the influences of variable SO42- and Fe2+ supplies on radial oxygen loss (ROL), iron plaque formation and As translocation in Avicennia marina upon exposure to As(III). The results indicate that A. marina is an As-tolerant plant, the application of iron and sulfur not only showed positive growth effects but also induced much higher amounts of ROL-induced iron plaque formation on root surfaces. The presence of iron plaque remarkably improved the proportion of As sequestration near this area but consequently reduced the proportion of As translocation in root. Therefore, it is concluded that iron plaque may act as a barrier for protection against As, and iron and sulfur play important roles in controlling the growth and translocation of As in A. marina seedlings.

Impact of epigallocatechin­3­gallate on expression of nuclear factor erythroid 2­related factor 2 and γ­glutamyl cysteine synthetase genes in oxidative stress­induced mouse renal tubular epithelial cells.

The aim of the present study was to investigate the antioxidant response mechanism of epigallocatechin­3­gallate (EGCG) in H2O2­induced mouse renal tubular epithelial cells (MRTECs). The cultured MRTECs were divided into normal, H2O2 (control) and EGCG treatment groups. The MTT assay was used to assess cell viability, and reverse transcription­quantitative polymerase chain reaction (RT­qPCR), immunocytochemical and western blot analyses were performed to detect the expression of nuclear factor erythroid 2­related factor 2 (Nrf2) and γ­glutamyl cysteine synthetase (γ­GCS). EGCG was able to mitigate H2O2­mediated cell damage. The RT­qPCR results demonstrated that EGCG was able to upregulate the gene expression of Nrf2 and γ­GCS in MRTECs in a dose­dependent manner. The immunocytochemistry and western blot analyses demonstrated that EGCG was able to increase the protein expression of Nrf2 and γ­GCS in MRTECs in a dose­dependent manner. Oxidative stress may lead to a decrease in the viability of MRTECs, while EGCG was able to promote the expression of Nrf2 and γ­GCS in MRTECs, thereby improving the antioxidant capacity of the cells and promoting the repair of oxidative stress injury.

A three-dimensional analysis of skeletal and dental characteristics in skeletal class III patients with facial asymmetry.

OBJECTIVE: To evaluate the skeletal and dental characteristics in skeletal class III patients with facial asymmetry and to analyse the relationships among various parts of the stomatognathic system to provide a theoretical basis for clinical practice. METHODS: Asymmetric cone-beam computed tomography data acquired from 56 patients were evaluated using Mimics 10.0 and 3-Matic software. Skeletal and dental measurements were performed to assess the three-dimensional differences between two sides. Pearson correlation analysis was used to determine the correlations among measurements. RESULTS: Linear measurements, such as ramal height, mandible body length, ramal height above the sigmoid notch (RHASN), maxillary height, condylar height, buccal and total cancellous bone thickness, and measurements of condylar size, were significantly larger on the nondeviated side than on the deviated side (P < 0.05). Crown root ratio and buccolingual angle of mandibular first molar were found to be significantly smaller on the nondeviated side than on the deviated side (P < 0.05). A negative correlation was also discovered between the buccolingual angle of mandibular first molar and the ramal height (P < 0.01). CONCLUSIONS: In patients with facial asymmetry, asymmetries in the mandible, maxilla and condylar morphology, and skeletal canting served as major components of skeletal asymmetry. Furthermore, a reduced thickness of buccal cancellous bone and a larger crown root ratio were found on the deviated side, indicating that orthodontic camouflage has limitations and potential risks. A combination of orthodontics and orthognathic surgery may be the advisable choice in patients with a menton deviation greater than 4 mm. An important association between vertical skeletal disharmony and dental compensation was also observed.

Effects of maxillary protraction appliances on airway dimensions in growing class III maxillary retrognathic patients: A systematic review and meta-analysis.

OBJECTIVES: The purpose of this study was to assess, through a systematic review and meta-analysis, the efficacy of maxillary protraction appliances (MPAs) on improving pharyngeal airway dimensions in growing class III patients with maxillary retrognathism. METHODS: An electronic search in PubMed, Cochrane Library, Web of Science, and EMBASE was until September 2nd, 2017. The assessments of methodological quality of the selected articles were performed using the Newcastle-Ottawa Scale. Review Manager 5.3 (provided by the Cochrane Collaboration) was used to synthesize the effects of MPAs on pharyngeal airway dimensions. RESULTS: Following full-text articles evaluation for eligibility, 6 studies (168 treated subjects and 140 untreated controls) were included in final quantitative synthesis and they were all high-quality. Compared to untreated control groups, the treatment groups had increased significantly nasopharyngeal airway dimensions with the following measurements: PNS-AD1 (fixed: mean difference, 1.33 mm, 95% CI, 0.48mm-2.19 mm, P = .002), PNS-AD2 (random: mean difference, 1.91 mm, 95% CI, 0.02mm-3.81 mm, P = .05), aerial nasopharyngeal area (fixed: mean difference, 121.91 mm2, 95% CI, 88.70 mm2-155.11 mm2, P < .00001) and total nasopharyngeal area (fixed: mean difference, 142.73 mm2, 95% CI, 107.90 mm2-177.56 mm2, P < .00001). Meanwhile, McNamara's upper pharynx dimension (fixed: mean difference, 0.96 mm, 95% CI, 0.29mm-1.63 mm, P = .005), which was highly related to post-palatal airway dimension, was also improved significantly. However, no statistically significant differences in adenoidal nasopharyngeal area (P > .05) and McNamara's lower pharynx dimension (P > .05) existed. CONCLUSIONS: MPAs can increase post-palatal and nasopharyngeal airway dimensions in growing skeletal class III subjects with maxillary retrusion. It may be suggested that MPAs have the potential to reduce the risk of obstructive sleep apnea syndrome in children with maxillary retrusion by enlarging airway space.

Calcium-sensing receptor-mediated mitogen-activated protein kinase pathway improves the status of transplanted mouse embryonic stem cells in rats with acute myocardial infarction.

Several studies have identified the critical role of calcium-sensing receptors (CaSRs) in cardiac ischaemia/reperfusion injury and cardiac hypertrophy and have demonstrated that CaSRs induce myocardial apoptosis by activating MAPKs. Using acute myocardial infarction rat models, we found that a combination therapy of CaSR inhibition and embryonic stem cell (ESC) transplantation after acute myocardial infarction (AMI) leads to a dramatic reduction in the infarct size; a significant increase in the maximum rising and falling rate (+dp/dtmax and -dp/dtmax, respectively) of left ventricular pressure; a significant decrease in left ventricular end-diastolic pressure; a significant decrease in the mRNA expression level of CaSR, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, p-ERK, p-JNK and p-P38 protein together with apoptosis indexes in the C and E groups; and a significant decrease in cTnT levels as well as LDH and CK activity. These findings indicate that cardiac function could be enhanced significantly by combination therapy with CaSR inhibition and ESC transplantation; the effect was better than ESC transplantation alone, and the mechanism might be associated with a reduction in cell apoptosis via the inhibition of the MAPK pathway. Apoptosis could be reduced through CaSR, which regulates the MAPK pathway and apoptosis-related protein. Our study indicated that CaSR inhibitors have a pivotal role in the treatment of AMI.

Protective effect of C4a against hyperoxic lung injury via a macrophage-dependent but not a neutrophil/lymphocyte-dependent signaling pathway.

Complement anaphylatoxins have been investigated extensively; however, the role of complement anaphylatoxin C4a in hyperoxic lung injury has yet to be investigated. To the best of our knowledge, the present study is the first to demonstrate the role of C4a in hyperoxic lung injury in vitro and in vivo. BALB/c mice were ventilated with 100% oxygen with or without C4a treatment for 36 h. The body weight and the relative lung weight of the mice were determined, along with any morphological changes in the lung. The expression levels of interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) were quantified in the lung tissue and bronchoalveolar lavage fluid (BALF) samples by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were determined using cytocentrifuge slides and a hemocytometer. Histamine release from total cells in the BALF was also analyzed. The relative mRNA expression levels of CD68, F4/80, CD64, CD19 and CD3 in the murine lung tissue were assessed by reverse transcription-quantitative polymerase chain reaction. The results revealed that hyperoxia induced lung injury and morphological changes, and increased the expression levels of IL-1, IL-6 and TNF-α, histamine release, the number of inflammatory cells, and the expression levels of CD68, F4/80, CD64, CD19 and CD3. The hyperoxia-induced morphological changes and inflammatory reaction were significantly attenuated in mice treated with C4a. Treatment with C4a also attenuated the increase in the total cell count, decreased the number of macrophages in the BALF, and suppressed the elevated mRNA expression levels of CD68 and F4/80 in the lung tissue samples. Conversely, treatment with C4a did not affect the number of neutrophils or lymphocytes in the BALF or the mRNA expression of CD64, CD19 and CD3 in lung tissue. In conclusion, C4a attenuated hyperoxic lung injury via a macrophage-dependent but not a neutrophil/lymphocyte-dependent pathway.

Effects of fucoidan on insulin stimulation and pancreatic protection via the cAMP signaling pathway in vivo and in vitro.

Diabetes is a global disease, in which pancreatic dysfunction is an important pathological process. In previous years, interest in the biological activities of seaweed has increased. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has been widely investigated. The present study aimed to determine the effects of fucoidan on insulin stimulation and pancreatic protection in vivo and in vitro. Goto­Kakizaki (GK) rats were provided with free access to standard food, with or without fucoidan, for 13 weeks, following which the body weights, and blood glucose and serum insulin levels of the rats were measured. Wistar rats were used as a control. In addition, the RIN­5F rat insulin­secreting cell line was treated with fucoidan in high glucose conditions, following which the dose­dependent and time­dependent effects of fucoidan were determined, and the concentration of insulin was measured. Glybenclamide was used as a positive control. In vivo, the body weight and serum insulin levels decreased, whereas blood glucose levels increased significantly in the GK rats, compared with the Wistar control rats. Although, fucoidan did not improve changes in body weight, the increased blood glucose levels were reduced and the decreased serum insulin levels were increased in the GK rats following oral administration of fucoidan. In vitro, fucoidan did not exhibit significant cytotoxicity towards the RIN­5F cells, and the insulin secretion increased significantly in a dose­ and time­dependent manner. Treatment with amylin, an islet amyloid polypeptide and glybenclamide inhibitor, did not inhibit the stimulatory activity of fucoidan. The results of the present study also demonstrated that the concentration of cyclic adenosine monophosphate (cAMP) was significantly increased in the fucoidan­treated RIN­5F cells, and this increase was dose­ and time­dependent. In addition, treatment with a phosphodiesterase inhibitor, which decreases the degradation of cAMP, significantly increased fucoidan­induced insulin secretion, whereas treatment with an adenylyl cyclase inhibitor, which decreases the generation of cAMP, significantly decreased fucoidan­induced insulin secretion. In conclusion, these data indicated that fucoidan may stimulate insulin secretion and provide pancreatic protection via the cAMP signaling pathway, in vivo and in vitro.

[Effect of adenovirus-mediated tissue factor pathway inhibitor gene transfer on apoptosis of rat vascular smooth muscle cells].

OBJECTIVE: Previous studies showed potential role of tissue factor pathway inhibitor (TFPI) on attenuating restenosis, we investigated the effect of TFPI gene transfer on vascular smooth muscle cells (VSMCs) apoptosis. METHODS: Human TFPI recombinant adenovirus or LacZ recombinant adenovirus or PBS were transferred to rat aortic VSMCs respectively in vitro. RT-PCR was used to detect the expression of exogenous TFPI gene. VSMCs were examined by cell counting and MTT. Apoptosis of VSMCs was detected by flow cytometry, TUNEL and electron microscope at different time after gene transfer. RESULTS: mRNA expression of TFPI was detected in VSMCs at the 3rd day after gene transfer. Cell numbers and absorbance value in Ad-TFPI group were similar as those in Ad-LacZ and PBS groups at the 1st, 3rd and 5th day but significantly lower at the 7th day (P<0.05) after gene transfer. The apoptosis rates in Ad-TFPI group tested by flow cytometry were all significant higher than those in Ad-lacZ groups at each time point. The positive rates in Ad-TFPI group determined by TUNEL were significant higher than those in Ad-LacZ groups at 3rd (10.82% +/- 1.57% vs. 3.46% +/- 0.93%), 5th and 7th (16.95% +/- 2.01% vs. 5.11% +/- 1.29%, all P<0.05) day post gene transfer. Electron microscope evidenced cell contracting, cytoplasm condensing, lightly swelled mitochondria, nucleus pyknosis and apoptotic body formation after gene transfer in Ad-TFPI group which were not shown in cells of LacZ and PBS groups. CONCLUSION: TFPI gene transfer could induce apoptosis in rat VSMCs which might be one of the mechanisms responsible for its beneficial effect on restenosis inhibition after angioplasty.