PCTR1 improves pulmonary edema fluid clearance through activating the sodium channel and lymphatic drainage in lipopolysaccharide-induced ARDS.

Acute respiratory distress syndrome (ARDS) is a lethal clinical syndrome characterized by damage of the epithelial barriers and accumulation of pulmonary edema fluid. Protectin conjugates in tissue regeneration 1 (PCTR1), an endogenously produced lipid mediator, are believed to exert anti-inflammatory and pro-resolution effects. PCTR1 (1 µg/kg) was injected at 8 hr after lipopolysaccharide (LPS; 14 mg/kg) administration, and the rate of pulmonary fluid clearance was measured in live rats at 1 hr after PCTR1 treatment. The primary type II alveolar epithelial cells were cultured with PCTR1 (10 nmol/ml) and LPS (1 µg/ml) for 8 hr. PCTR1 effectively improved pulmonary fluid clearance and ameliorated morphological damage and reduced inflammation of lung tissue, as well as improved the survival rate in the LPS-induced acute lung injury (ALI) model. Moreover, PCTR1 markedly increased sodium channel expression as well as Na, K-ATPase expression and activity in vivo and in vitro. In addition, PCTR1i also upregulated the expression of LYVE-1 in vivo. Besides that, BOC-2, HK7, and LY294002 blocked the promoted effect of PCTR1 on pulmonary fluid clearance. Taken together, PCTR1 upregulates sodium channels' expression via activating the ALX/cAMP/P-Akt/Nedd4-2 pathway and increases Na, K-ATPase expression and activity to promote alveolar fluid clearance. Moreover, PCTR1 also promotes the expression of LYVE-1 to recover the lymphatic drainage resulting in the increase of lung interstitial fluid clearance. In summary, these results highlight a novel systematic mechanism for PCTR1 in pulmonary edema fluid clearance after ALI/ARDS, suggesting its potential role in a therapeutic approach for ALI/ARDS.

Changes in growth performance, aflatoxin B1 residues, immune response and antioxidant status of Litopenaeus vannamei fed with AFB1-contaminated diets and the regulating effect of dietary myo-inositol supplementation.

This study aimed to investigate if myo-inositol (MI) supplementation could alleviate adverse effects caused by aflatoxin B1 (AFB1) with respect to growth performance, AFB1 residues, immune response and antioxidant status of Litopenaeus vannamei. 800 shrimp (initial weight: 1.1 g) were divided into five groups: MI0 (basal diet); MI0 + LA, MI0 + HA, MI200 + LA and MI200 + HA fed with AFB1-contaminated diets (LA, low concentration AFB1; HA, high concentration AFB1; MI200, adding 200 mg MI kg-1 diet). The results showed that HA significantly decreased growth performance, systemic inositol content and lipid content. AFB1 residues were detected in the hepatopancreas of shrimp, but not the muscle. Histological lesions were observed in MI0 + LA and MI0 + HA groups. HA supplementation raised malondialdehyde and protein carbonyl content and reduced some antioxidant enzyme activities and immune-related genes expression, which was slightly ameliorated by MI supplementation. Our results suggest that myo-inositol may slightly mitigate negative impacts caused by AFB1 in L. vannamei.

Maresin Conjugates in Tissue Regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury.

Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage-derived sulfido-conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up-regulating epithelial sodium channel (ENaC) and Na+ -K+ -adenosine triphosphatase (Na, K-ATPase) expressions in vivo. MCTR1 also activated Na, K-ATPase and elevated phosphorylated-Akt (P-Akt) by up-regulating the expression of phosphorylated Nedd4-2 (P-Nedd4-2) in vivo and in vitro. However, BOC-2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS).

MCTR1 alleviates lipopolysaccharide-induced acute lung injury by protecting lung endothelial glycocalyx.

Endothelial glycocalyx degradation, critical for increased pulmonary vascular permeability, is thought to facilitate the development of sepsis into the multiple organ failure. Maresin conjugates in tissue regeneration 1 (MCTR1), a macrophage-derived lipid mediator, which exhibits potentially beneficial effects via the regulation of bacterial phagocytosis, promotion of inflammation resolution, and regeneration of tissue. In this study, we show that MCTR1 (100 ng/mouse) enhances the survival of mice with lipopolysaccharide (LPS)-induced (15 mg/kg) sepsis. MCTR1 alleviates LPS (10 mg/kg)-induced lung dysfunction and lung tissue inflammatory response by decreasing inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß [IL-1ß], and IL-6) expression in serum and reducing the serum levels of heparan sulfate (HS) and syndecan-1. In human umbilical vein endothelial cells (HUVECs) experiments, MCTR1 (100 nM) was added to the culture medium with LPS for 6 hr. MCTR1 treatment markedly inhibited HS degradation by downregulating heparanase (HPA) protein expression in vivo and in vitro. Further analyses indicated that MCTR1 upregulates sirtuin 1 (SIRT1) expression and decreases NF-κB p65 phosphorylation. In the presence of BOC-2 or EX527, the above effects of MCTR1 were abolished. These results suggest that MCTR1 protects against LPS-induced sepsis in mice by attenuating pulmonary endothelial glycocalyx injury via the ALX/SIRT1/NF-κB/HPA pathway.

Effects of dietary hydrolyzed yeast (Rhodotorula mucilaginosa) on growth performance, immune response, antioxidant capacity and histomorphology of juvenile Nile tilapia (Oreochromis niloticus).

An 8 weeks feeding experiment was conducted to evaluate the effects of dietary supplementation with hydrolyzed yeast (HY) (Rhodotorula mucilaginosa) on growth performance, hematological parameters, immune response and antioxidant ability of juvenile Nile tilapia. Five isonitrogenous and isolipidic diets (32% protein and 4% lipid) with different levels (0%, 0.125%, 0.25%, 0.5%, 1%) of HY were formulated. Each diet was randomly assigned to quadruplicate groups of fish (initial body weight 19.1 ±â€¯0.01 g). Results indicated that significantly higher specific growth rate (SGR) and lower feed conversion rate (FCR) were obtained in fish fed 1% HY diet than that of fish fed 0% HY diet (P < 0.05). Fish fed 0.25% HY diet showed the lowest value of hepatopancreas somatic indices (HSI) and significantly lower than that of fish fed 0% HY diet (P < 0.05). Meanwhile, protein and ash in the whole-body content of fish fed 1% HY diet was significantly higher than that of fish fed 0%-0.5% HY diets. Serum immunological parameters showed that the lysozyme (LZM) activity and Complement C3 content were significantly increased by dietary supplementation of 0.5%-1% HY (P < 0.05). However, dietary supplementation with 0.125%-1% HY significantly decreased the activity of myeloperoxidase (MPO) (P < 0.05). Antioxidant status in serum and liver was significantly enhanced by dietary supplementation of 0.25%-1% HY through the remarkably improved superoxide dismutase (SOD) activity both in serum and liver, the raised total antioxidative capacity (T-AOC) of serum as well as the notably reduced malondialdehyde (MDA) content in the liver (P < 0.05). However, T-AOC in the liver was not significantly influenced among all diet treatments (P > 0.05). Villi height and intraepithelial lymphocytes (IEFs) of mid-intestine were significantly higher in fish fed 0.5%-1% HY diets (P < 0.05). The challenge test demonstrated the enhanced protection against Streptococcus iniae strain by the obtained higher cumulative survival rate. In conclusion, dietary supplementation of 1% HY could maintain the better growth performance, nutrient composition as well as immune response and antioxidant capacity for juvenile Nile tilapia.

Maresin1 Alleviates Metabolic Dysfunction in Septic Mice: A 1H NMR-Based Metabolomics Analysis.

Maresin1 (MaR1), a new anti-inflammatory and proresolving lipid mediator, has been proven to exert organ-protective effects in septic animal models. However, the potential mechanisms are still not fully elucidated. In this study, we sought to explore the impact of MaR1 on metabolic dysfunction in cecal ligation and puncture- (CLP-) induced septic mice. We found that MaR1 significantly increased the overall survival rate and attenuated lung and liver injuries in septic mice. In addition, MaR1 markedly reduced the levels of proinflammatory cytokines (TNF-α and IL-6) and alleviated mitochondrial damage. Based on a 1H NMR-based metabolomics analysis, CLP-induced septic mice had increased levels of acetate, pyruvate, and lactate in serum and decreased levels of alanine, aspartate, glutamate, and fumarate in lungs. However, these metabolic disorders, mainly involving energy and amino acid metabolism, can be recovered by MaR1 treatment. Therefore, our results suggest that the protective effects of MaR1 on sepsis could be related to the recovery of metabolic dysfunction and the alleviation of inflammation and mitochondrial damage.

Detoxification and immunoprotection of Zn(II)-curcumin in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxin B1.

Aflatoxins, which was produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing or storage, could cause severe health problems and reduction of yield during shrimp cultures. To evaluate toxic effects of aflatoxin B1 (AFB1) in juvenile Pacific white shrimp (Litopenaeus vannamei) and potential protective effect of Zn(II)-curcumin (Zn-CM), four experimental diets (control, 500 µg/kg AFB1, 500 µg/kg AFB1+100 mg/kg Zn-CM, 500 µg/kg AFB1+200 mg/kg Zn-CM) were formulated in quadruplicate to feed the shrimp for 8 weeks. The results revealed that AFB1 could induce significant decrease in final body weight (FBW), weight gain (WG, %) and visible variations of the hepatopancreas structures in L.vannamei. Compared with AFB1 group, AFB1+100 mg/kg Zn-CM group significantly ameliorated the toxic effects of AFB1 on growth performance, while AFB1+100 mg/kg Zn-CM group had no effect on growth performance. Dietary AFB1+100 mg/kg Zn-CM enhanced phenoloxidase (PO) (P < 0.05) activity. Both dietary AFB1+100 mg/kg Zn-CM and AFB1+200 mg/kg Zn-CM reduced inducible nitric oxide synthase (iNOS) activity and glutathione (GSH) level, decreased the content of malondialdehyde (MDA) (P < 0.05) in hepatopancreas compared with AFB1 group. Transmission electron microscopy (TEM) analysis demonstrated that Zn-CM could relieve the microvilli transformation and mitochondria accumulation reduction caused by AFB1. Consequently, the results demonstrated that suitable Zn-CM could mitigate the AFB1-induced hepatotoxicity and immunotoxicity effects on L.vannamei.

Reversal of the Apoptotic Resistance of Non-Small-Cell Lung Carcinoma towards TRAIL by Natural Product Toosendanin.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers cancer cell death via its association with death receptors on the cell membrane, but exerts negligible side effects on normal cells. However, some non-small-cell lung carcinoma (NSCLC) patients exhibited resistance to TRAIL treatment in clinical trials, and the mechanism varies. In this study, we described for the first time that toosendanin (TSN), a triterpenoid derivative used in Chinese medicine for pain management, could significantly sensitize human primary NSCLC cells or NSCLC cell lines to TRAIL-mediated apoptosis both in vitro and in vivo, while showing low toxicity against human primary cells or tissues. The underlying apoptotic mechanisms involved upregulation of death receptor 5 (DR5) and CCAAT/enhancer binding protein homologous protein, which is related to the endoplasmic reticulum stress response, and is further associated with reactive oxygen species generation and Ca2+ accumulation. Surprisingly, TSN also induced autophagy in NSCLC cells, which recruited membrane DR5, and subsequently antagonized the apoptosis-sensitizing effect of TSN. Taken together, TSN can be used to sensitize tumors and the combination of TRAIL and TSN may represent a useful strategy for NSCLC therapy; moreover, autophagy serves as an important drug resistance mechanism for TSN.

High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences.

The Coleoptera (beetles) exhibits tremendous morphological, ecological, and behavioral diversity. To better understand the phylogenetics and evolution of beetles, we sequenced three complete mitogenomes from two families (Cleridae and Meloidae), which share conserved mitogenomic features with other completely sequenced beetles. We assessed the influence of six datasets and three inference methods on topology and nodal support within the Coleoptera. We found that both Bayesian inference and maximum likelihood with homogeneous-site models were greatly affected by nucleotide compositional heterogeneity, while the heterogeneous-site mixture model in PhyloBayes could provide better phylogenetic signals for the Coleoptera. The amino acid dataset generated more reliable tree topology at the higher taxonomic levels (i.e. suborders and series), where the inclusion of rRNA genes and the third positions of protein-coding genes improved phylogenetic inference at the superfamily level, especially under a heterogeneous-site model. We recovered the suborder relationships as (Archostemata+Adephaga)+(Myxophaga+Polyphaga). The series relationships within Polyphaga were recovered as (Scirtiformia+(Elateriformia+((Bostrichiformia+Scarabaeiformia+Staphyliniformia)+Cucujiformia))). All superfamilies within Cucujiformia were recovered as monophyletic. We obtained a cucujiform phylogeny of (Cleroidea+(Coccinelloidea+((Lymexyloidea+Tenebrionoidea)+(Cucujoidea+(Chrysomeloidea+Curculionoidea))))). This study showed that although tree topologies were sensitive to data types and inference methods, mitogenomic data could provide useful information for resolving the Coleoptera phylogeny at various taxonomic levels by using suitable datasets and heterogeneous-site models.

Effect of cadmium-polluted diet on growth, salinity stress, hepatotoxicity of juvenile Pacific white shrimp (Litopenaeus vannamei): Protective effect of Zn(II)-curcumin.

Cadmium (Cd) is one of the major transitional metals that have toxic effects on aquatic organisms. To investigate the effects of dietary cadmium on growth, salinity stress, hepatotoxicity in juvenile Pacific white shrimp (L. vannamei) and potential protective effect of Zn(II)-curcumin, five experimental diets (control, 100mg/kg Zn(II)-curcumin, 30mg/kg Cd, 30mg/kg Cd+100mg/kg Zn(II)-curcumin, 30mg/kg Cd+200mg/kg Zn(II)-curcumin) were formulated. The results showed that Cd at 30mg/kg induced significant increase in weight gain, specific growth rate and visible alterations to the hepatopancreas structures of L. vannamei. Compared with control diet, 100mg/kg Zn(II)-curcumin added diet had no effect on growth performance or feed utilization, while healthier hepatopancreas and less plasma ALT, AST production was found. Moreover, 200mg/kg dietary Zn(II)-curcumin significantly ameliorated the Cd induced hepatotoxicity while 100mg/kg dietary Zn(II)-curcumin slightly ameliorated. Cd accumulation in the whole body was decreasing and Metallothioneins like was increasing in hepatopancreas with increasing dietary Zn(II)-curcumin level. The shrimp fed with dietary Zn(II)-curcumin showed higher survival rate after acute salinity change. Therefore, it can be demonstrated that hepatotoxicity and hormesis could be induced by Cd when Cd levels were 30mg/kg, Zn(II)-curcumin could mitigate the effects of dietary Cd on L. vannamei.

Contribution of CFTR to alveolar fluid clearance by lipoxin A4 via PI3K/Akt pathway in LPS-induced acute lung injury.

The lipoxins are the first proresolution mediators to be recognized and described as the endogenous "braking signals" for inflammation. We evaluated the anti-inflammatory and proresolution bioactions of lipoxin A4 in our lipopolysaccharide (LPS-)induced lung injury model. We demonstrated that lipoxin A4 significantly improved histology of rat lungs and inhibited IL-6 and TNF- α in LPS-induced lung injury. In addition, lipoxin A4 increased alveolar fluid clearance (AFC) and the effect of lipoxin A4 on AFC was abolished by CFTRinh-172 (a specific inhibitor of CFTR). Moreover, lipoxin A4 could increase cystic fibrosis transmembrane conductance regulator (CFTR) protein expression in vitro and in vivo. In rat primary alveolar type II (ATII) cells, LPS decreased CFTR protein expression via activation of PI3K/Akt, and lipoxin A4 suppressed LPS-stimulated phosphorylation of Akt. These results showed that lipoxin A4 enhanced CFTR protein expression and increased AFC via PI3K/Akt pathway. Thus, lipoxin A4 may provide a potential therapeutic approach for acute lung injury.

[Clinical characteristic analysis of 96 cases of hypersensitivity pneumonitis].

OBJECTIVE: To improve understanding of the clinical characteristics and diagnosis of hypersensitivity pneumonitis (HP). METHODS: We retrospectively analyzed the clinical data, including clinical symptoms, laboratory tests, exposure, pulmonary function tests, chest CT imaging and cytological classification of bronchoalveolar lavage (BAL) of 96 patients with HP from Jan 2001 to Jun 2011 in Peking Union Medical College Hospital. We divided the patients into 2 groups: a pathologically-confirmed group and a clinically-suspected group. RESULTS: There were 58 females and 41 males. The median age at the diagnosis was 53 years. The most common exposures were low-molecular-weight chemicals (42.7%) and animal proteins (37.5%). Common clinical symptoms included dyspnea on exertion (90.6%) and cough (76.0%). Pulmonary function test showed diffusion abnormality (73.5%) and restrictive ventilatory impairment (59.7%). Chest CT scan revealed patchy or diffuse bilateral ground-glass opacities (64.6%), centrilobular nodules (21.9%), and air trapping (15.6%). Reticulation (45.8%), traction bronchiectasis (21.9%) and honeycombing(9.4%) were present in chronic HP. BAL lymphocyte counts > 0.2 and CD4/CD8 < 0.9 were more commonly seen in patients with a disease course of less than 1 year. The pathologically-confirmed group and the clinically-suspected group shared many similar characteristics including age at diagnosis, gender, clinical manifestation, pulmonary function impairments and imaging findings, but significant differences existed in certain parameters. In the pathologically- confirmed group, the duration of disease was longer (24 months vs 6 months, Z = -2.492, P = 0.013) and clubbed fingers were more common (23.4% vs 8.2%, χ(2) = 4.227, P = 0.040). Diffusion abnormality was present in more patients of this group (90.7% vs 44.0%, χ(2) = 35.219, P < 0.01). By CT scan, reticulation, traction bronchiectasis and honeycombing (57.5% vs 26.5%, χ(2) = 9.434, P < 0.01) were more evident as compared to the clinically-suspected group. The value of transbronchial lung biopsy for diagnosing HP was limited, with a positive result of only 8.2%. Surgical lung biopsy was needed in uncertain cases. CONCLUSION: The diagnosis of HP was difficult. In some cases a clinical diagnosis can be made by combination of history of exposure, CT manifestations and cell classification of BAL. For atypical cases a multi-disciplinary approach including pathologists, radiologists and pulmonologists is needed.

Effect of dietary vitamin E and selenium supplementation on growth, body composition, and antioxidant defense mechanism in juvenile largemouth bass (Micropterus salmoides) fed oxidized fish oil.

Six oxidized fish oil contained diets were formulated to investigate the effect of graded levels of vitamin E (V(E)) (α-tocopherol acetate: 160, 280, and 400 mg kg(-1)) associated with either 1.2 or 1.8 mg kg(-1) selenium (Se) on growth, body composition, and antioxidant defense mechanism of juvenile largemouth bass. Another control diet containing fresh fish oil with 160 mg kg(-1) V(E) and 1.2 mg kg(-1) Se was also prepared. Over a 12-week feeding trial, about 5 % of Micropterus salmoide fed diet OxSe1.2/V(E)160 showed inflammation and hemorrhage symptoms at the base of dorsal, pectoral, and tail fin. Fish in all treatments survived well (above 90 %). Feed intakes (88.42-89.58 g fish(-1)) of all treatments were comparable. Growth performances (weight gain and specific growth rate) and feed utilization (feed and protein efficiency ratio) were significantly impaired by dietary oil oxidation, and they did not benefit from neither V(E) nor Se supplementation. Regardless of dietary V(E) and Se supplementation, oxidized oil ingestion resulted in markedly decreased hepatosomatic index and intraperitoneal fat ratio. Oxidized oil ingestion also induced markedly lower liver and muscle lipid contents, and these effects could be alleviated by dietary Se supplementation. Dietary oil oxidation stimulated hepatic catalase activities relative to the control, and supplementation of V(E) abrogated this effect. Hepatic reduced glutathione content in the control was markedly higher than that of treatment OxSe1.2/V(E)160, without any significant differences comparing with the other oxidized oil receiving groups. Hepatic glutathione peroxidase activity and liver Se concentration reflected dietary Se profile, whereas liver V(E) level reflected dietary V(E) profile. Compared with the control, fish fed diet OxSe1.2/V(E)160 obtained markedly higher serum, liver and muscle malondialdehyde contents, which droppe significantly with increasing either V(E) or Se supplementation. In conclusion, the overall results in this study suggested that both V(E) and Se inclusion could protect largemouth bass from the oxidative damage challenged by dietary oil oxidation.

FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

Fused in Sarcoma (FUS) proteinopathy is a feature of frontotemporal lobar dementia (FTLD), and mutation of the fus gene segregates with FTLD and amyotrophic lateral sclerosis (ALS). To study the consequences of mutation in the fus gene, we created transgenic rats expressing the human fus gene with or without mutation. Overexpression of a mutant (R521C substitution), but not normal, human FUS induced progressive paralysis resembling ALS. Mutant FUS transgenic rats developed progressive paralysis secondary to degeneration of motor axons and displayed a substantial loss of neurons in the cortex and hippocampus. This neuronal loss was accompanied by ubiquitin aggregation and glial reaction. While transgenic rats that overexpressed the wild-type human FUS were asymptomatic at young ages, they showed a deficit in spatial learning and memory and a significant loss of cortical and hippocampal neurons at advanced ages. These results suggest that mutant FUS is more toxic to neurons than normal FUS and that increased expression of normal FUS is sufficient to induce neuron death. Our FUS transgenic rats reproduced some phenotypes of ALS and FTLD and will provide a useful model for mechanistic studies of FUS-related diseases.

[Evaluation on HIV antibody testing strategy currently used in China].

OBJECTIVE: To evaluate the yield of HIV antibody testing strategy currently used on different populations, in China. METHODS: (1) The following samples were collected and tested according to the currently used HIV antibody testing strategy in China. 103 133 samples from the general populations (outpatients, new recruits and blood donors), 1276 people under high risk (spouses of the HIV infected individuals, intravenous drug users) and 2323 biochemical or immunological abnormal samples. (2) Retrospective analysis was done on data from the HIV testing among outpatients in General Hospital of People's Armed Police Forces, from Jan., 2002 to Dec., 2008 and in three provincial central HIV test and confirmatory laboratories. RESULTS: (1) The yields of HIV antibody screening were significantly different in different populations. The probability of screening reactive to be true positive was 50% in high risk population, significantly higher than in the general population. The probability of screening reactive to be true positive was 19.58% in the confirmatory laboratory mainly towards the general population, but significantly lower than results from the confirmatory laboratories done on the high risk population. (2) From 2002 to 2008, in the General Hospital of People's Armed Police Forces, the probability of screening reactive to be true positive in the clinical HIV test was increasing from 3.7% to 16.0%, where as the efficiency of the repeat screening testing decreased from 92.6% to 61.5%. CONCLUSION: The predictive value of HIV antibody screening reactive was significantly greater in high risk population than in general population. The precision of HIV antibody initial screening was substantially increased with the improvement of HIV antibody test kits and of quality control in the HIV test laboratories in recent years. It is suggested that different HIV test strategies to be implemented in different populations.

Very low density lipoprotein receptor subtype II silencing by RNA interference inhibits cell proliferation in hepatoma cell lines.

Very low density lipoprotein receptor (VLDLR) belongs to the low density lipoprotein receptor family, it is divided into two subtypes according to forms with an absence (type II) or a presence (type I) of the O-linked sugar domain. VLDLR have been detected in kinds of cancers so far; however, the subtype of VLDLR in hepatocellular carcinoma (HCC) tissues and hepatoma cell lines has yet to be reported. We detected the VLDLR expression in 39 cases of hepatocellular carcinoma and in three kinds of hepatoma cell lines: HepG2, HBV transfected HepG2.2.15, SMMC-7721 and normal human fetal liver cell line LO2 using RT-PCR and western blotting. The results showed that both type I and type II VLDLR were detected in HCC tissues and hepatoma cell lines, and the type II VLDLR expression was significantly higher than that of type I in cell lines. We inhibited the type II VLDLR expression by shRNA-mediated RNA interference in HepG2, SMMC-7721 cell and then subsequently found the cell proliferation slowed down. The cyclinD1 expression confirmed the cell cycle was arrested at the G0/G1 phase, suggesting that inhibiting the type II VLDLR expression may have a positive impact on carcinogenesis of HCC.

[Clinical analysis of four cases of primary ciliary dyskinesia and literature review].

OBJECTIVE: To study the clinical characteristics and diagnosis of primary ciliary dyskinesia (PCD). METHODS: Four cases diagnosed as PCD by cilia electron microscopy examination from Jan. 2007 to Aug. 2009 in this hospital were retrospectively analyzed, and the related literature was reviewed. RESULTS: In the 4 patients, there were 1 male and 3 females. The age at disease onset was 0 - 10 years, and the age at diagnosis was 15 - 53 years. The most common symptoms were productive cough (4/4), sinusitis (3/4), and shortness of breath (3/4). Other manifestations included situs inversus (2/4), infertility (1/4), and tympanitis (1/4). Hypoxemia was found in 3 cases. Obstructive ventilatory impairment accompanied with diffusion dysfunction was observed in 2 cases, while the pulmonary function tests were normal in the other 2 cases. All 4 cases received chest CT scan, and bronchiectasis was present in all of them. Bilateral diffuse micronodules and patchy infiltrates were found in 3 and 2 cases respectively. Electron microscopic examination of the endobronchial biopsy specimen showed lack of dynein arms in 4, lack of muco-cilia in 2, and abnormal arrangement of microtubules in 2 cases. CONCLUSIONS: The Kartagener syndrome is relatively easy to be diagnosed, because it is characterized by the triad of sinusitis, bronchiectasis and situs inversus. However, PCD without situs inversus often goes unrecognized. PCD should be considered in patients with childhood onset disease, bronchiectasis, centrilobular micronodules or tree-in-bud signs in CT scan. Examination of the ciliary ultrastructure is essential to the confirmation of the diagnosis.

The phox domain of sorting nexin 5 lacks phosphatidylinositol 3-phosphate (PtdIns(3)P) specificity and preferentially binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2).

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 A resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new alpha2'-helix and a much longer alpha3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.

Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.

Neuritic retraction represents a prominent feature of the degenerative phenotype associated with mutations in leucine rich repeat kinase 2 (LRRK2) that are implicated in autosomal dominant and some cases of sporadic Parkinson's disease. Alterations in macroautophagy, the vacuolar catabolism of cytoplasmic constituents, have been described in Parkinson's disease. In this study, we utilized retinoic-acid differentiated SH-SY5Y cells to determine whether autophagy contributes to mutant LRRK2-associated neurite degeneration. Transfection of pre-differentiated SH-SY5Y cells with LRRK2 cDNA containing the common G2019S mutation resulted in significant decreases in neurite length, which were not observed in cells transfected with wild type LRRK2 or its kinase-dead K1906M mutation. G2019S LRRK2 transfected cells also exhibited striking increases in autophagic vacuoles in both neuritic and somatic compartments, as demonstrated by fluorescence and western blot analysis of the autophagy marker green fluorescent protein-tagged microtubule-associated protein Light Chain 3 and by transmission electron microscopy. RNA interference knockdown of LC3 or Atg7, two essential components of the conserved autophagy machinery, reversed the effects of G2019S LRRK2 expression on neuronal process length, whereas rapamycin potentiated these effects. The mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) reduced LRRK2-induced neuritic autophagy and neurite shortening, implicating MAPK/ERK-related signaling. These results indicate an active role for autophagy in neurite remodeling induced by pathogenic mutation of LRRK2.

Dileucine motif is sufficient for internalization and synaptic vesicle targeting of vesicular acetylcholine transporter.

Efficient cholinergic transmission requires accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs). However, the signals that regulate this vesicular targeting are not well characterized. Although previous studies suggest that the C-terminus of the transporter is required for its SV targeting, it is not clear whether this region is sufficient for this process. Furthermore, a synaptic vesicle-targeting motif (SVTM) within this sequence remains to be identified. Here we use a chimeric protein, TacA, between an unrelated plasma membrane protein, Tac, and the C-terminus of VAChT to demonstrate the sufficiency of the C-terminus for targeting to synaptic vesicle-like vesicles (SVLVs) in PC12 cells. TacA shows colocalization and cosedimentation with the SV marker synaptophysin. Deletion mutation analysis of TacA demonstrates that a short, dileucine motif-containing sequence is required and sufficient to direct this targeting. Dialanine mutation analysis within this sequence suggests indistinguishable signals for both internalization and SV sorting. Using additional chimeras as controls, we confirm the specificity of this region for SVLVs targeting. Therefore, we suggest that the dileucine-containing motif is sufficient as a dual signal for both internalization and SV targeting during VAChT trafficking.