Network pharmacology analysis and experimental verification of the antitumor effect and molecular mechanism of isocryptomerin on HepG2 cells.

Isocryptomerin (ISO) is a flavonoid isolated from the natural medicine Selaginellae Herba, which has various pharmacological activities. This study investigated the antitumor effect and underlying molecular mechanism of ISO on hepatocellular carcinoma (HCC) HepG2 cells. The cell viability assay revealed that ISO has a considerable killing effect on HCC cell lines. The apoptosis assay showed that ISO induced mitochondria-dependent apoptosis through the Bad/cyto-c/cleaved (cle)-caspase-3/cleaved (cle)-PARP pathway. The network pharmacological analysis found 13 key target genes, and epidermal growth factor receptor (EGFR), AKT, mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling pathways were strongly associated with ISO against HCC. Further verification of the results showed that ISO induced apoptosis by increasing p-p38 and p-JNK expression and decreasing p-EGFR, p-SRC, p-ERK, and p-STAT3 expression. Furthermore, ISO induced G0/G1 phase arrest by downregulating p-AKT, Cyclin D, and CDK 4 expression and upregulating p21 and p27 expression in HepG2 cells. Moreover, ISO inhibited HepG2 cell migration by decreasing p-GSK-3ß, ß-catenin, and N-cadherin expression and increasing E-cadherin expression. Additionally, ISO promoted ROS accumulation in HepG2 cells, and ISO-induced apoptosis, arrest cell cycle, and inhibition of migration were reversed by an ROS scavenger, N-acetyl- l-cysteine. Overall, ISO induced cell apoptosis and cell cycle arrest and inhibited cell migration by ROS-mediated EGFR, AKT, and MAPK signaling pathways in HepG2 cells.

2-(Naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone induces apoptosis via ROS-mediated MAPK, AKT, and STAT3 signaling pathways in HepG2 human hepatocellular carcinoma cells.

1,4-naphthoquinone and its derivatives have attracted widespread attention due to their multiple biological activities, such as induction of cancer cell apoptosis; however, most of these compounds have high cytotoxicity. In this study, in order to reduce their toxicity and increase their potential anti-tumor effects, we synthesized a novel 1,4-naphthoquinone derivative named 2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone (NTDMNQ), and investigated its apoptotic effects and underlying mechanism. Our results showed that NTDMNQ inhibited the viability of HepG2, Hep3B, and Huh7 human hepatocellular carcinoma (HCC) cells. It also increased the accumulation of cells in the G0/G1 phase of the cell cycle by increasing the expression levels of p-p53, p21 and p27, while decreasing the levels of Cyclin D1, Cyclin E, Cyclin-dependent kinase 2 (CDK2), CDK4, and CDK6. Inhibition of reactive oxygen species (ROS) by the ROS scavenger N-acetyl-L-cysteine (NAC) decreased apoptosis in NTDMNQ-treated cells. Western blot analysis showed that NTDMNQ increased the phosphorylation of p38 and c-Jun N-terminal kinase (JNK), and decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and signal transducer and activator of transcription-3 (STAT3); these effects were blocked by NAC. Both the JNK inhibitor (SP600125) and p38 inhibitor (SB203580) reversed the phosphorylation of STAT3, and the ERK inhibitor (FR180204) and AKT inhibitor (LY294002) reduced the expression of STAT3. Taken together, these findings suggest that NTDMNQ induces apoptosis via ROS-mediated MAPK, AKT and STAT3 signaling pathways in HepG2 cells, and may be a potent anticancer agent.

Atractylodin Induces Apoptosis and Inhibits the Migration of A549 Lung Cancer Cells by Regulating ROS-Mediated Signaling Pathways.

Atractylodin (ATR) has anticancer effects on some tumor cells by inducing apoptosis, but its mechanism in lung cancer remains unclear. This study investigates the inhibitory effect of ATR on A549 lung cancer cells. Cell viability was detected by the Cell Counting Kit-8 assay, and results showed that ATR could significantly inhibit the proliferation of A549 cells. Apoptosis was detected by Annexin V-FITC/PI staining, and apoptosis rate and mitochondrial membrane potential were detected by flow cytometry. Results showed that the effect of ATR on the apoptosis of A549 cells was negatively correlated with the change in mitochondrial membrane potential. Western blot analysis showed that ATR regulated apoptosis induced by mitogen-activated protein kinase, signal transducer and activator of transcription 3, and nuclear factor kappa B signaling pathways. Analyses of reactive oxygen species (ROS), cell cycle, and cell migration showed that ATR induced intracellular ROS accumulation as an initiation signal to induce cell cycle arrest regulated by the AKT signaling pathway and cell migration inhibition regulated by the Wnt signaling pathway. Results showed that ATR can inhibit cell proliferation, induce cell apoptosis, induce cell cycle arrest, and inhibit the migration of A549 cells (p < 0.05 was considered statistically significant, * p < 0.05, ** p < 0.01 and *** p < 0.001).

Malaria from hyperendemicity to elimination in Hekou County on China-Vietnam border: an ecological study.

BACKGROUND: Malaria control and elimination are challenged by diversity and complexity of the determinants on the international border in the Great Mekong Sub-region. Hekou, a Chinese county on the China-Vietnam border, was used to document Chinese experiences and lessons for malaria control and elimination. METHODS: The design was an ecological study. Malaria burden before 1951 and procedures of 64 years (1952-2015) from malaria hyperendemicity to elimination are described. Single and bilinear regression analysis was utilized to analyse the relationship between the annual malaria incidence (AMI) and gross domestic product (GDP), urbanization rate, and banana planting area (BPA). RESULTS: There was a huge malaria burden before 1951. AMI was reduced from 358.62 per 1000 person-years in 1953 to 5.69 per 1000 person-years in 1960. A system of primary health services, comprising three levels of county township hospitals and village health stations maintained malaria control and surveillance activities in changing political and social-economic settings. However, potential under-reported of malaria and market-oriented healthcare led to a malaria epidemic in 1987. Strong political commitment reoriented malaria from a control to an elimination programme. High coverage of malaria intervention and population access to intervention was crucial for malaria control and elimination; meanwhile, AMI was closely associated with socio-economic development, correlation coefficients (R) -0.6845 (95% CI -0.7978, -0.6845) for national GDP, -0.7014 (-0.8093, -0.7014) for national urbanization rate and -0.5563 (-0.7147, -0.3437) for BPA. CONCLUSIONS: Multifactor, including political commitment, effective interventions, social and economic development and changing ecological environment, and the complicated interactions between these factors contribute to malaria elimination in Hekou County.

[Genome-wide identification and analyses of 4CL gene families in Pyrus bretschneideri Rehd].

4-coumaric acid: coenzyme A ligase (4CL) gene is one of the key genes involved in the regulation of lignin metabolism and the synthesis of flavonoid and other secondary metabolites in plant, while the synthesized and polymerized lignin is deposited in cell walls and leads to thickening of secondary walls in some parenchyma cells and formation of stone cells. To better understand the variety and quantity of 4CL genes in Pyrus bretschneideri Rehd., we used the amino acid and cDNA databases of Pyrus bretschneideri Rehd. genome to screen 4CL gene family, and analyzed their classification, evolutionary relationships, physical location, gene structure and conserved motif. Our results showed that 29 4CL genes were identified and preliminarily characterized, and these 4CL genes were distributed in all chromosomes except chromosomes 4, 8, 11, 12 and clustered on chromosomes 9 and 17 through gene location analysis. The relationship between 4CL gene structure and evolution was further determined by comparing gene structure and phylogenetic tree. These findings provide a basis for further analysis of 4CL gene function in Pyrus bretschneideri Rehd.

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways.

Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3ß, N-cadherin, and ß-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.

Value of red blood cell distribution width in prediction of diastolic dysfunction in cirrhotic cardiomyopathy.

BACKGROUND: Clinical diagnosis of cirrhotic cardiomyopathy (CCM) often encounters challenges of lack of timeliness and disease severity, with the commonly positive indicator usually associated with advanced heart failure. AIM: To explore suitable biomarkers for early CCM prediction. METHODS: A total of 505 eligible patients were enrolled in this study and divided into four groups according to Child-Pugh classification: Group I, Class A without CCM (105 cases); Group II, Class A with CCM (175 cases); Group III, Class B with CCM (139 cases); and Group IV, Class C with CCM (86 cases). Logistic regression and receiver operating characteristic (ROC) curve analyses were performed to determine whether red blood cell distribution width (RDW) was an independent risk factor for CCM risk. The relationships between RDW and Child-Pugh scores, Model for End-Stage Liver Disease (MELD) scores, and N-terminal pro-brain natriuretic peptide (NT-proBNP) were analyzed by Pearson correlation analysis. RESULTS: A constant RDW increase was evident from Group I to Group IV (12.54 ± 0.85, 13.29 ± 1.19, 14.30 ± 1.96, and 16.25 ± 2.13, respectively). Pearson correlation analysis showed that RDW was positively correlated with Child-Pugh scores (r = 0.642, P < 0.001), MELD scores (r = 0.592, P < 0.001), and NT-proBNP (r = 0.715, P < 0.001). Furthermore, between Group I and Group II, RDW was the only significant index (odds ratio: 2.175, 95% confidence interval [CI]: 1.549-3.054, P < 0.001), and it reached statistical significance when examined by ROC curve analysis (area under the curve: 0.686, 95%CI: 0.624-0.748, P < 0.001). CONCLUSION: RDW can serve as an effective and accessible clinical indicator for the prediction of diastolic dysfunction in CCM, in which a numerical value of more than 13.05% may indicate an increasing CCM risk.

Characterization of Prototheca zopfii associated with outbreak of bovine clinical mastitis in herd of Beijing, China.

Prototheca zopfii (P. zopfii) has become an important cause of bovine mastitis in many countries. In the present study, to better understand the occurrence of one clinical mastitis (CM) outbreak due to P. zopfii, the molecular characterization and resistance patterns of the microalga were described. P. zopfii strains were isolated from 17 of 23 quarters, which suffered CM in the outbreak, and 7 of 46 CM recovered quarters before the outbreak, as well as 2 of 75 environmental samples in the dairy farm. All strains were identified as genotype 2 by genotype-specific PCR analysis. Results of in vitro antimicrobial and antifungal susceptibility tests indicated that these strains were resistant to majority of tested drugs, with the only exception of amphotericin B, nystatin, streptomycin, gentamicin, and amikacin. This is the first report about CM outbreak caused by P. zopfii in China. These data suggest that P. zopfii may represent a serious risk in the studied herd, and this microalga could be an important potential pathogen causing mastitis in dairy herds of Beijing.

Characterization of light chain c-terminal extension sequence variant in one bispecific antibody.

Protein modifications such as post-translational modifications (PTMs) and sequence variants (SVs) occur frequently during protein biosynthesis and have received great attention by biopharma industry and regulatory agencies. In this study, an aberrant peak near light chain (LC) was observed in the non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS) electrophoretogram during cell line development of one bispecific antibody (BsAb) product, and the detected mass was about 944 Da higher than LC. The corresponding peak was then enriched by denaturing size-exclusion chromatography (SEC-HPLC) and further characterized by nrCE-SDS and peptide mapping analyses. De novo mass spectra/mass spectra (MS/MS) analysis revealed that the aberrant peak was LC related sequence variant, with the truncated C-terminal sequence "SFNR" ("GEC"deleted) linked with downstream SV40 promotor sequence "EAEAASASELFQ". The unusual sequence was further confirmed by comparing with the direct synthetic peptide "SFNREAEAASASELFQ". It was demonstrated by mRNA sequencing of the cell pool that the sequence variant was caused by aberrant splicing at the transcription step. The prepared product containing this extension variant maintained well-folded structure and good functional properties though the LC/Heavy chain (HC) inter-chain disulfide was not formed. Several control strategies to mitigate the risk of this LC related sequence variant were also proposed.

[The protective effects of bifidobacterial adhesin on ischemic reperfusion injury of intestine in rats].

OBJECTIVE: To investigate the protection effect of bifidobacterial adhesin for intestine ischemia/reperfusion (I/R) injury on gut barrier function in rat. METHODS: Seventy-two male SD rats were randomly divided into sham operation group (n = 24), I/R model group (n = 24) and pretreatment group of bifidobacterial adhesin (pretreatment group, n = 24). Six rats were anatomized at 6 h, 1 d, 4 d and 7d after inducing I/R model in each group, respectively. The pathological changes of the terminal ilea and the blood levels of TNFα, IL-6, IL-10, diamine oxidase (DAO), and the activity and content of D-lactic acid were observed. RESULTS: The blood levels of TNFα, IL-6, DAO and D-lactic acid in I/R model group were significantly higher than sham operation group at all time points (P < 0.05), while the blood level of IL-10 was no significantly change. The activity of IL-6 and DAO in pretreatment group was significantly lower than I/R model group at all time points (P < 0.05), the blood level of TNFα in pretreatment group was significantly lower than I/R model group at 1 d, the blood level of D-lactic was significantly lower than I/R model group at 4 d and 7 d (P < 0.05). Intestinal pathological damages were obviously milder in pretreatment group than I/R model group at all time points (Chiu's pathological scores: 6 h, 3.22 ± 0.22 vs 3.57 ± 0.20; 1 d, 3.77 ± 0.13 vs 3.90 ± 0.12; 4 d, 2.93 ± 0.23 vs 3.07 ± 0.21; 7 d, 2.10 ± 0.30 vs 2.22 ± 0.17, all P < 0.05). CONCLUSION: The pretreatment of bifidobacterial adhesin could protect the intestinal mucosa from I/R injury, and alleviate intestinal ischemic reperfusion injury.

[Evaluation of shensongyangxin capsules in the treatment of paroxysmal atrial fibrillation: a randomized, double-blind and controlled multicenter trial].

OBJECTIVE: To evaluate the efficacy and safety of Chinese medicinal shensongyangxin capsules in the treatment of paroxysmal atrial fibrillation. METHODS: From August 2007 to July 2008, Beijing Chaoyang Hospital conducted a multicenter study, select the eleven hospital's outpatient subjects, aged 18 to 75 years old, male or female, paroxysmal atrial fibrillation (at least one electrocardiogram diagnosis) seizure frequency ≥ 2 times/month, according to the ratio 1:1:1, subjects were randomly divided into three groups: a. shensongyangxin group, taking shensongyangxin capsule 4 + propafenone analogues 150 mg, 3 times a day; b. propafenone group, taking propafenone tablets 150 mg + 4 shensongyangxin analogues, 3 times a day; shensongyangxin capsule + propafenone group, taking shensongyangxin capsule 4 + propafenone 150 mg, 3 times a day. The treatment course is 8 weeks, with 3 times of follow-up. RESULTS: Total of 349 cases of paroxysmal atrial fibrillation, which 117 cases in shensongyangxin group, 115 cases in propafenone group; 117 cases in shensongyangxin + propafenone group. The baseline data analysis showed that there were no significantly difference (P > 0.05) among the three groups of atrial fibrillation seizure frequency, vital signs, general condition, medical history, 24-hour ambulatory ECG, 12-lead normal electrocardiogram, cardiac ultrasound and symptoms. The comparison before and after (8 weeks) treatment showed that the frequency (from 6 times/m to 2 times/m in each group, P < 0.01), number of cases [from 46 (43.3%) to 22 (20.8%), 43 (43.4%) to 25 (25.3%), and 40 (40.6%) to 31 (29.2%), respectively P < 0.01] and duration time of attack of atrial fibrillation (from 4 h to 0.5 h, 4 h to 0.5 h, and 4.25 h to 0.5 h, respectively P < 0.01) all decreased in three groups. No significant difference among the three groups comparing the overall effect (62.3%, 58.6%, and 58.5%, respectively, P > 0.05), while the efficacy of TCM symptoms in shensongyangxin group (80.2%) was better than that of propafenone group (67.7%) (P < 0.05). Safety evaluation showed that adverse reaction rate was 1.8% in shensongyangxin group, and 8.2% and 5.4% in propafenone group and shensongyangxin + propafenone group. CONCLUSION: Shensongyangxin capsules and propafenone have comparable efficacies in the treatment of PAF. The efficacy of TCM symptoms is better than propafenone. Shensongyangxin capsules have an excellent profile of safety.

Clinical efficacy and safety of tirofiban combined with conventional dual antiplatelet therapy in ACS patients undergoing PCI.

Challenges remain for clinicians over balancing the efficacy of active antithrombotic therapy and simultaneous bleeding reduction in patients. The clinical data of 347 patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) were retrospectively analyzed. On the basis of the given tirofiban, the patients were assigned into three different dose groups: high dose group (group H), medium dose group (group M), and low dose group (group L). The tirofiban efficacy was evaluated in terms of major adverse cardiovascular event (MACE) parameters and lab endpoints, including platelet count and function. The tirofiban safety was assessed by the occurrence of bleeding events. The patients were followed up for 1 month after the PCI. No significant difference in MACE events was evident among these groups (p > 0.05). Groups H and M reported an obvious reduction in platelet count (p < 0.05 for both) and an increased platelet inhibition rate (p < 0.05 for both). Group H showed a higher rate of total bleeding events than the other groups (Group H vs. Group M: 34.4% vs. 16.5%; Group H vs. Group L: 34.4% vs. 10.3%; p < 0.05 for both). A proper administration of a low dose of tirofiban may be a superior alternative in treating ACS patients, which can produce a similar favorable clinical outcome and a decrease in bleeding complication.

Protein oxidation implicated as the primary determinant of bacterial radioresistance.

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

Hepatitis E virus re-infection accelerates hepatocellular carcinoma development and relapse in a patient with liver cirrhosis: A case report and review of literature.

BACKGROUND: Hepatitis E virus (HEV) superinfection is a suspected promoting factor for hepatocellular carcinoma (HCC) in patients with chronic hepatitis and cirrhosis. However, to date, very few cases of HEV-related HCC have been reported. Nevertheless, the role of HEV re-infection in cirrhotic liver without other chronic hepatitis infections has rarely been explored. CASE SUMMARY: A 53-year-old male farmer was diagnosed with liver cirrhosis and splenomegaly in August 2016, accompanied with negative HEV-IgM and positive HEV-IgG. No evidence of hepatitis B virus or hepatitis C virus infection was found. Since then the patient was evaluated for liver function and viral parameters every 3 mo. In June 2017, the patient presented severe fatigue with whole body itching and was diagnosed with HCC. Afterwards this patient experienced quick HCC development, progression, relapse, and metastasis in the following 8 mo, and presented persistent dual positivity of HEV-IgM and HEV-IgG. This patient had a long history of smoking and alcohol consumption. CONCLUSION: This unique case invokes the importance of HEV surveillance and treatment among cirrhotic patients, HCC cases, and blood donors.

Liquiritin inhibits proliferation and induces apoptosis in HepG2 hepatocellular carcinoma cells via the ROS-mediated MAPK/AKT/NF-κB signaling pathway.

Liquiritin (LIQ), a major constituent of Glycyrrhiza Radix, exhibits various pharmacological activities. In this study, to explore the potential anti-cancer effects and its underlying molecular mechanisms of LIQ in hepatocellular carcinoma (HCC) cells. LIQ significantly decreased viability and induced apoptosis in HepG2 cells by decreasing mitochondrial membrane potential and regulating Bcl-2 family proteins, cytochrome c, cle-caspase-3, and cle-PARP. The cell cycle analysis and western blot analysis revealed that LIQ induced G2/M phase arrest through increased expression of p21 and decreased levels of p27, cyclin B, and CDK1/2. The flow cytometry and western blot analysis also suggested that LIQ promoted the accumulation of ROS in HepG2 cells and up-regulated the phosphorylation expression levels of p38 kinase, c-Jun N-terminal kinase (JNK), and inhibitor of NF-κB (IκB-α); the phosphorylation levels of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), signal transducer activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) were down-regulated. However, these effects were reversed by N-acetyl-L-cysteine (NAC), MAPK, and AKT inhibitors. The findings demonstrated that LIQ induced cell cycle arrest and apoptosis via the ROS-mediated MAPK/AKT/NF-κB signaling pathway in HepG2 cells, and the LIQ may serve as a potential therapeutic agent for the treatment of human HCC.

Cytisine exerts anti-tumour effects on lung cancer cells by modulating reactive oxygen species-mediated signalling pathways.

Cytisine is a natural product isolated from plants and is a member of the quinolizidine alkaloid family. This study aims to investigate the effect of cytisine in human lung cancer. Cell viability was determined using the CCK-8 assay, and the results showed that cytisine inhibited the growth of lung cancer cell lines. The apoptotic effects were evaluated using flow cytometry, and the results showed that cytisine induced mitochondrial-dependent apoptosis through loss of the mitochondrial membrane potential; increased expression of BAD, cleaved caspase-3, and cleaved-PARP; and decreased expression levels of Bcl-2, pro-caspase-3, and pro-PARP. In addition, cytisine caused G2/M phase cell cycle arrest that was associated with inhibiting the AKT signalling pathway. During apoptosis, cytisine increased the phosphorylation levels of JNK, p38, and I-κB, and decreased the phosphorylation levels of ERK, STAT3, and NF-κB. Furthermore, cytisine treatment led to the generation of ROS, and the NAC attenuated cytisine-induced apoptosis. In vivo, cytisine administration significantly inhibited the lung cancer cell xenograft tumorigenesis. In conclusion, cytisine plays a critical role in suppressing the carcinogenesis of lung cancer cells through cell cycle arrest and induction of mitochondria-mediated apoptosis, suggesting that it may be a promising candidate for the treatment of human lung cancer.

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3 (glycoprotein IIb/IIIa). PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family. OBJECTIVES: To determine the molecular basis of GT and characterize the mutation by in vitro expression studies. RESULTS: Analysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of beta3, exposed on her platelet surface, but a complete absence of alphaIIbbeta3. Sequence analysis revealed a novel C470A transversion in exon 4 of the alphaIIb gene predicting a Pro126His alteration in the blade 2 of the alphaIIb beta propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alphaIIb and wild-type beta3 failed to express alphaIIbbeta3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature alphaIIb. Immunoprecipitation with antibody against beta3 demonstrated pro-alphaIIb in the cells expressing the mutant alphaIIbbeta3, indicating pro-alphaIIbbeta3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-alphaIIbbeta3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker. CONCLUSIONS: A novel Pro126His mutation in alphaIIb compromised transport of the pro-alphaIIbbeta3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired alphaIIbbeta3 transport is responsible for the thrombasthenia in this patient.

Higher serum lectin-like oxidized low-density lipoprotein receptor-1 in patients with stable coronary artery disease is associated with major adverse cardiovascular events: A multicentre pilot study.

INTRODUCTION: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the pathophysiology of atherosclerosis and acute coronary syndromes (ACS). Circulating soluble LOX-1 (sLOX-1) has been linked to the risk of coronary artery disease (CAD). Our aim was to test if baseline serum sLOX-1 was associated with major adverse cardiovascular events (MACE) in patients with stable CAD. MATERIALS AND METHODS: This multicentre pilot study enrolled 833 stable CAD patients. All patients were followed for two years. Serum sLOX-1 concentrations were detected by enzyme-linked immunosorbent assay (ELISA). The association between sLOX-1 concentrations and MACE was assessed by logistic regression, Kaplan-Meier survival curves and Cox proportional hazards analyses. Logistic regression analysis was employed to assess the predictors of complex lesion. RESULTS: Multivariate logistic regression analysis revealed that sLOX-1 concentration was an independent predictor of MACE (OR 2.07, 95%CI 1.52 - 2.82; P < 0.001). Kaplan-Meier cumulative survival curves showed that the incidence of MACE in patients with a high sLOX-1 concentration was significantly higher than in patients with an intermediate or low sLOX-1 concentration (P < 0.001). Soluble LOX-1 concentrations were independently correlated with coronary complex lesions (OR 2.32, 95%CI 1.81 - 2.97; P < 0.001). CONCLUSIONS: Baseline sLOX-1 concentrations were correlated with 2-year MACE in stable CAD patients. Furthermore, patients with high serum sLOX-1 concentrations had higher cumulative incidence of MACE compared to those with low serum sLOX-1 concentrations.

Baseline Serum sLOX-1 Concentrations Are Associated with 2-Year Major Adverse Cardiovascular and Cerebrovascular Events in Patients after Percutaneous Coronary Intervention.

BACKGROUND: Soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) may be a potential biomarker of coronary artery disease (CAD) and stroke. OBJECTIVE: We aimed to investigate the association and prognostic value of elevated sLOX-1 concentrations with regard to long-term major adverse cardiovascular and cerebrovascular events (MACCEs) in patients with CAD undergoing primary percutaneous coronary intervention (PCI). METHODS: A total of 1011 patients were enrolled. Serum sLOX-1 concentrations were detected by the enzyme-linked immunosorbent assay (ELISA). Patients were followed for 2 years. Multivariate Cox regression and Kaplan-Meier survival curve were explored to assess the association between sLOX-1 and MACCEs. A receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of sLOX-1. RESULTS: Two-year MACCEs were associated with serum sLOX-1 concentrations (HR 1.278, 95% CI 1.019-1.604, P = 0.034), left main disease (HR 2.938, 95% CI 1.246-6.925, P = 0.014), small-caliber stents used (HR 2.207, 95% CI 1.189-4.095, P = 0.012), and total stent length (HR 1.057, 95% CI 1.005-1.112, P = 0.030). Serum sLOX-1 concentration ≥ 1.10 ng/ml had maximum sensitivity and specificity in predicting the occurrence of 2-year MACCEs (P < 0.001). Patients with higher serum sLOX-1 concentrations showed a significantly higher incidence of MACCEs in the Kaplan-Meier curve (P < 0.001). The combination of any of the risk factors identified in multiple Cox regression was associated with a stepwise increase in MACCE rate (P < 0.001). CONCLUSIONS: High baseline serum sLOX-1 concentration predicts 2-year MACCEs and shows an additional prognostic value to conventional risk factors in patients after primary PCI. sLOX-1 determination might play a complementary role in the risk stratification of patients with CAD treated with PCI.

Utilization of DNA as a sole source of phosphorus, carbon, and energy by Shewanella spp.: ecological and physiological implications for dissimilatory metal reduction.

The solubility of orthophosphate (PO4(3-)) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca2+-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca2+ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca2+-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5' nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2',3' cyclic nucleotide 2' phosphodiesterase/3' nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.