An extracellular vesicle microRNA-initiated 3D DNAzyme motor for colorectal cancer diagnosis.

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual-parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three-dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in the free state or in extracellular vesicles. Quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA-initiated cleavage of fluorescent substrate strands by the DNAzyme motors. The practicability was systematically validated with microRNA-21-5p and microRNA-10b-5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets at 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a series of extracellular vesicles from clinical samples, which exhibited good distinguishability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system has potential to be easily adapted by minor alteration of the DNA sequences and fluorophore sets for detection of multiple miRNAs and even other types of biomarkers such as proteins. Therefore, it shows promise to be widely applied in various fields such as early diagnosis of cancer and its prognostic assessment.

Effects of norepinephrine­induced activation of rat vascular adventitial fibroblasts on proliferation and migration of BMSCs involved in vascular remodeling.

Vascular remodeling caused by vascular injury such as hypertension and atherosclerosis is a complex process involving a variety of cells and factors, and the mechanism is unclear. A vascular injury model was simulated by adding norepinephrine (NE) to culture medium of vascular adventitial fibroblasts (AFs). NE induced activation and proliferation of AFs. To investigate the association between the AFs activation and bone marrow mesenchymal stem cells (BMSCs) differentiation in vascular remodeling. BMSCs were cultured with supernatant of the AFs culture medium. BMSC differentiation and migration were observed by immunostaining and Transwell assay, respectively, while cell proliferation was measured using the Cell Counting Kit-8. Expression levels of smooth muscle actin (α-SMA), TGF-ß1 and SMAD3 were measured using western blot assay. The results indicated that compared with those in the control group, in which BMSCs were cultured in normal medium, expression levels of α-SMA, TGF-ß1 and SMAD3 in BMSCs cultured in medium supplemented with supernatant of AFs, increased significantly (all P<0.05). Activated AFs induced the differentiation of BMSCs into vascular smooth muscle-like cells and promoted proliferation and migration. AFs activated by NE may induce BMSCs to participate in vascular remodeling. These findings may help design and develop new approaches and therapeutic strategies for vascular injury to prevent pathological remodeling.

Effect of the serum 25-hydroxyvitamin D level on risk for short-term residual dizziness after successful repositioning in benign paroxysmal positional vertigo stratified by sex and onset age.

Objective: A low serum 25-hydroxyvitamin D (25(OH)D) level is relevant to both the occurrence and recurrence of benign paroxysmal positional vertigo (BPPV). However, whether it also contributes to residual dizziness (RD) after successful repositioning maneuvers is unknown. Therefore, this study aimed to explore the correlation between the serum 25(OH)D level and short-term RD severity in patients with BPPV after successful repositioning maneuvers. Methods: In total, 251 patients with BPPV after successful repositioning were enrolled and prospectively followed up for 1 week (W1). Serum 25(OH)D values were detected by chemiluminescence immunoassay at enrollment (W0). In addition, we explored the relationship between 25(OH)D values at baseline and RD severity at W1 in different subgroups stratified by sex and onset age (early-onset, ≤50 years; late-onset, >50 years). Results: The serum 25(OH)D level of female patients was significantly lower than that of male patients (15.9 ± 6.8 vs. 19.8 ± 6.6 ng/ml, p < 0.001). Its level also decreased in early-onset patients compared to late-onset ones (15.3 ± 5.9 vs. 18.0 ± 7.3 ng/ml, p = 0.003). In addition, early-onset female patients had lower 25(OH)D values than late-onset female patients (14.0 ± 5.5 vs. 17.1 ± 7.2 ng/ml, p = 0.004). However, this difference was not observed between early- and late-onset male patients. Among early-onset female patients, the 25(OH)D values of the moderate-to-severe RD group were lower than those of the minor or no RD group (10.9 ± 3.3 vs. 14.7 ± 5.7 vs. 15.0 ± 5.9 ng/ml, p = 0.046). Multivariate analysis found that decreased 25(OH)D values were related to the occurrence of moderate-to-severe RD in early-onset female patients (OR = 0.801; p = 0.022). This effect did not exist in late-onset female or male patients with BPPV. Conclusions: Age and sex differences in serum 25(OH)D levels exist in patients with BPPV. A decreased 25(OH)D level in early-onset female patients may increase the odds of moderate-to-severe RD 1 week after successful repositioning maneuvers.

Clinical and cVEMP Evaluation Predict Short-Term Residual Dizziness After Successful Repositioning in Benign Paroxysmal Positional Vertigo.

Objective: Residual dizziness (RD) is a frequent symptom with unknown pathogenesis, often complained about by the patients with benign paroxysmal positional vertigo (BPPV), even after a successful canalith repositioning procedure (CRP). This study aims to quantitatively evaluate the short-term RD severity and its risk factors in patients with BPPV after successful CRPs. Methods: In total two hundred and twenty patients with BPPV after successful CRPs (W0) were prospectively followed up for 1 week (W1). Besides demographics and serial neuropsychological assessments (including dizziness handicap inventory-DHI, etc.), patients also received cervical/ocular vestibular evoked myogenic potential (c/oVEMP) evaluation. RD was defined as patients with dizziness or imbalance, dizziness visual analog scale (VAS) >1, and without positional vertigo or nystagmus at W1. Demographic, clinical, and VEMPs differences were compared among the three groups: patients with minor (dizziness VAS 1-3) and moderate-to-severe RD (dizziness VAS > 3) and without RD. Results: The total frequency of RD at W1 was 49.1% (n = 108), with 32.3% (n = 71) minor, and 16.8% (n = 37) moderate-to-severe RD. Logistic regression analyses revealed that RD was closely associated with DHI status (OR = 2.101, P = 0.008) at W0, this effect was not present for minor RD. In addition to DHI score > 30 (OR = 4.898, P < 0.001) at W0, bilateral cVEMP absence (OR = 4.099, P = 0.005) was also an independent influential factor of moderate-to-severe RD. Conclusion: Our study highlights the importance of RD quantified evaluation. DHI score >30 and bilateral cVEMP absence could increase the risk of short-term moderate-to-severe RD.

Identifying Quantitative Trait Loci and Candidate Genes Conferring Resistance to Soybean Mosaic Virus SC7 by Quantitative Trait Loci-Sequencing in Soybean.

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, R SMV -11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

Geography-Driven Evolution of Potato Virus A Revealed by Genetic Diversity Analysis of the Complete Genome.

Potato virus A (PVA), a member of the genus Potyvirus, is an important potato pathogen that causes 30%-40% yield reduction to global potato production. Knowledge on the genetic structure and the evolutionary forces shaping the structure of this pathogen is limited but vital in developing effective management strategies. In this study, we investigated the population structure and molecular evolution of PVA by analyzing novel complete genomic sequences from Chinese isolates combined with available sequences from Europe, South America, Oceania, and North America. High nucleotide diversity was discovered among the populations studied. Pairwise F ST values between geographical populations of PVA ranged from 0.22 to 0.46, indicating a significant spatial structure for this pathogen. Although purifying selection was detected at the majority of polymorphic sites, significant positive selection was identified in the P1, NIa, and NIb proteins, pointing to adaptive evolution of PVA. Further phylogeny-trait association analysis showed that the clustering of PVA isolates was significantly correlated with geographic regions, suggesting that geography-driven adaptation may be an important determinant of PVA diversification.

Walking stability in patients with benign paroxysmal positional vertigo: an objective assessment using wearable accelerometers and machine learning.

BACKGROUND: Benign paroxysmal positional vertigo (BPPV) is one of the most common peripheral vestibular disorders leading to balance difficulties and increased fall risks. This study aims to investigate the walking stability of BPPV patients in clinical settings and propose a machine-learning-based classification method for determining the severity of gait disturbances of BPPV. METHODS: Twenty-seven BPPV outpatients and twenty-seven healthy subjects completed level walking trials at self-preferred speed in clinical settings while wearing two accelerometers on the head and lower trunk, respectively. Temporo-spatial variables and six walking stability related variables [root mean square (RMS), harmonic ratio (HR), gait variability, step/stride regularity, and gait symmetry] derived from the acceleration signals were analyzed. A support vector machine model (SVM) based on the gait variables of BPPV patients were developed to differentiate patients from healthy controls and classify the handicapping effects of dizziness imposed by BPPV. RESULTS: The results showed that BPPV patients employed a conservative gait and significantly reduced walking stability compared to the healthy controls. Significant different mediolateral HR at the lower trunk and anteroposterior step regularity at the head were found in BPPV patients among mild, moderate, and severe DHI (dizziness handicap inventory) subgroups. SVM classification achieved promising accuracies with area under the curve (AUC) of 0.78, 0.83, 0.85 and 0.96 respectively for differentiating patients from healthy controls and classifying the three stages of DHI subgroups. Study results suggest that the proposed gait analysis that is based on the coupling of wearable accelerometers and machine learning provides an objective approach for assessing gait disturbances and handicapping effects of dizziness imposed by BPPV.

Structural and Functional Changes in the Cerebellum and Brainstem in Patients with Benign Paroxysmal Positional Vertigo.

Benign paroxysmal positional vertigo (BPPV) is one of the most common peripheral vestibular diseases. Since the peripheral vestibular system connects with the cerebellum via the brainstem, repeated episodic vertigo may result in progressive structural and functional changes in the cerebellum and brainstem. In the present work, voxel-based morphometry (VBM) of T1-weighted images and resting-state functional magnetic resonance imaging (fMRI) in 32 patients with BPPV and 32 matched healthy controls were used to assess cerebellar and brainstem anatomical and spontaneous resting-state brain activity alterations associated with BPPV. We used a spatially unbiased infratentorial template toolbox in combination with VBM to analyze cerebellar and brainstem gray matter volume (GMV), fractional amplitude of low-frequency fluctuations (fALFF), and regional homogeneity (ReHo). Patients with BPPV showed decreased GMV in the right cerebellum posterior lobe/cerebellar tonsil extending to the cerebellum anterior lobe and pons relative to healthy controls. BPPV patients also exhibited significantly higher fALFF values in the right pons and left pons and higher ReHo values in the left cerebellum posterior lobe/Crus2 than the controls. Furthermore, the fALFF z-scores in the pons were positively correlated with the duration of vertigo at baseline and dizziness visual analog scale scores 1 week after canalith repositioning procedures (CRPs). BPPV patients exhibited structural and functional changes in the cerebellum and pons, which may reflect the adaptation and plasticity of these anatomical structures after repeated attacks of episodic vertigo. These results indicate that the changes in pons function may be closely related to residual dizziness after CRPs.

Tetramethylpyrazine alleviates acute kidney injury by inhibiting NLRP3/HIF­1α and apoptosis.

The aim of the present study was to investigate the protective effect and underlying mechanism of tetramethylpyrazine (TMP) on renal ischemia reperfusion injury (RIRI) in rats, which refers to the injury caused by the restoration of blood supply and reperfusion of the kidney after a period of ischemia. Sprague­Dawley rats were randomly divided into a Sham group, renal ischemia­reperfusion (I/R) group and TMP group. TMP hydrochloride (40 mg/kg, 6 h intervals) was given via intraperitoneal injection immediately after reperfusion in the TMP group, after 24 h the kidney tissues were taken for follow­up experiments. Pathological changes in the kidney tissues were observed by periodic acid­Schiff staining. Renal function was assessed by measuring levels of serum creatinine and blood urea nitrogen, and inflammatory cytokines tumor necrosis factor (TNF)­α and interleukin (IL)­6. Renal cell apoptosis was detected by TUNEL­DAPI double staining, mRNA and protein changes were analyzed by reverse transcription­quantitative PCR and western blotting. Cell viability was measured using a CCK­8 assay. It was found that the renal tissues of the sham operation group were notably abnormal, and the renal tissues of the I/R group were damaged, while the renal tissues of the TMP group were less damaged compared with those of the I/R group. Compared with the I/R group, the serum creatinine and blood urea nitrogen levels in the TMP group were low (all P<0.05), levels of inflammatory cytokines TNF­α and IL­6 decreased, the apoptotic rate was low (all P<0.05), and the relative expression levels of nucleotide­oligomerization domain­like receptor 3 (NLRP3) protein and mRNA in renal tissues were low (all P<0.05). The expression levels of hypoxia­inducible factor 1­α and NLRP3 increased after oxygen and glucose deprivation (OGD), and reduced after treatment with OGD and TMP (all P<0.05). It was concluded that TMP can reduce renal injury and improve renal function in RIRI rats, and its mechanism may be related to the reduction of NLRP3 expression in renal tissues.

Identification of potential crucial genes and molecular mechanisms in glioblastoma multiforme by bioinformatics analysis.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor of the adult central nervous system and is associated with poor prognosis. The present study aimed to identify the hub genes in GBM in order to improve the current understanding of the underlying mechanism of GBM. The RNA­seq data were downloaded from The Cancer Genome Atlas database. The edgeR package in R software was used to identify differentially expressed genes (DEGs) between two groups: Glioblastoma samples and normal brain samples. Gene Ontology (GO) functional enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed using Database for Annotation, Visualization and Integrated Discovery software. Additionally, Cytoscape and Search Tool for the Retrieval of Interacting Genes/Proteins tools were used for the protein­protein interaction network, while the highly connected modules were extracted from this network using the Minimal Common Oncology Data Elements plugin. Next, the prognostic significance of the candidate hub genes was analyzed using UALCAN. In addition, the identified hub genes were verified by reverse transcription­quantitative (RT­q) PCR. In total, 1,483 DEGs were identified between GBM and control samples, including 954 upregulated genes and 529 downregulated genes (P<0.01; fold­change >16) and these genes were involved in different GO terms and signaling pathways. Furthermore, CDK1, BUB1, BUB1B, CENPA and GNG3 were identified as key genes in the GBM samples. The UALCAN tool verified that higher expression level of CENPA was relevant to poorer overall survival rates. In conclusion, CDK1, BUB1, BUB1B, CENPA and GNG3 were found to be potential biomarkers for GBM. Additionally, 'cell cycle' and 'γ­aminobutyric acid signaling' pathways may serve a significant role in the pathogenesis of GBM.

Seasonal variation of idiopathic benign paroxysmal positional vertigo correlates with serum 25-hydroxyvitamin D levels: a six-year registry study in Shanghai, China.

Seasonal variation of benign paroxysmal positional vertigo (BPPV) occurrence has been reported in recent years. Whether the seasonality of BPPV also exists in Chinese patients and whether it correlates with serum vitamin D levels is unexplored. We retrospectively analyzed the data of 1269 new-onset idiopathic BPPV patients registered in our vertigo outpatient clinic over a six-year period. Additionally, serum 25-hydroxyvitamin D levels during this period were measured in 877 patients by chemiluminescence immunoassay. We delineated the changing trend of the monthly BPPV patient numbers and serum 25-hydroxyvitamin D levels, and the correlation between them was explored. December to next March is the top 4 months with higher BPPV patient numbers. The median BPPV patient numbers in winter group were higher than those in summer group (20 vs. 16 patients, p < 0.05). Median 25-hydroxyvitamin D levels in winter group were much lower than those in summer group (16.3 vs. 20.8 ng/ml, p < 0.001) and autumn group (16.3 vs. 19.3 ng/ml, p < 0.05). A moderate negative correlation was observed between median serum 25-hydroxyvitamin D levels and BPPV patient numbers each month. The onset of BPPV also shows a seasonal fluctuation in Chinese patients. This phenomenon may be related to serum vitamin D levels.

Molecular Evolutionary Analysis of Potato Virus Y Infecting Potato Based on the VPg Gene.

Potato virus Y (PVY) is an important plant pathogen infecting solanaceous crops, causing significant losses to global potato and tobacco production. Some aspects of the plant pathology and molecular biology of PVY have been studied intensively, but the evolutionary dynamics of this virus are poorly understood. Here, we performed a comprehensive set of rigorous evolutionary analyses using 177 nucleotide sequences of the viral genome linked protein (VPg) gene, which interacts with the plant eukaryotic translation initiation factor 4E (eIF4E). Our Bayesian analysis reveals that the VPg gene of PVY has been evolving at a rate of 5.60 × 10-4 subs/site/year (95% credibility interval 3.35 × 10-4-8.17 × 10-4), which is equivalent to those of other plant-infecting RNA viruses. We identified different evolutionary constraints on the two clades of PVY, clade N and clade O, whose diverge time were estimated at the year 1861 CE (95% credibility interval 1750-1948 CE). We also found that genetic variations were correlated with geographic regions, suggesting that the evolution of this pathogen is strongly affected by geographical associated factors. Taken together, the results of our study have potential implications for the control strategies of PVY.

Apocynin inhibited NLRP3/XIAP signalling to alleviate renal fibrotic injury in rat diabetic nephropathy.

AIMS: In this animal study, we tried to test the hypothesis that apocynin could play an anti-inflammation role by inhibiting NLRP3/X-linked inhibitor of apoptosis protein (XIAP) signalling and have an effect on antifibrosis in rats with diabetic nephropathy. MAIN METHODS: Diabetic nephropathy rats were induced by tail-vein injection of streptozotocin at 60 mg/kg body weight in sodium citrate buffer (0.01 M, pH 4.5) with unrestricted access to food/water for 12 weeks, and rats with blood glucose levels above 18.0 mM were considered diabetic; the damage index for glomerular mesangial cells damage index was calculated by morphological examinations; protein and mRNA changes were analysed by western blotting immunohistochemistry and real-time quantitative polymerase chain reaction; interstitial fibrosis was assessed and scored using Masson's staining. KEY FINDINGS: In rats with diabetic nephropathy, apocynin (1) reduced renal injury and improved renal function; (2) downregulated the expression of NLRP3 in renal cortex; (3) downregulated the expression of XIAP in renal cortex; and (4) attenuated renal fibrosis. SIGNIFICANCE: As an inhibitor of reactive oxygen species (ROS), apocynin could downregulate the expression of NLRP3 and XIAP, and alleviate renal fibrosis, which meant not only that ROS was one type of ligands of NLRP3, but also that ROS mechanism and NLRP3 activation might be therapeutic targets in the treatment of diabetic nephropathy in the future.

Validation of 5-item and 2-item questionnaires in Chinese version of Dizziness Handicap Inventory for screening objective benign paroxysmal positional vertigo.

As possible candidate screening instruments for benign paroxysmal positional vertigo (BPPV), studies to validate the Dizziness Handicap Inventory (DHI) sub-scale (5-item and 2-item) and total scores are rare in China. From May 2014 to December 2014, 108(55 with and 53 without BPPV) patients complaining of episodic vertigo in the past week from a vertigo outpatient clinic were enrolled for DHI evaluation, as well as demographic and other clinical data. Objective BPPV was subsequently determined by positional evoking maneuvers under the record of optical Frenzel glasses. Cronbach's coefficient α was used to evaluate the reliability of psychometric scales. The validity of DHI total, 5-item and 2-item questionnaires to screen for BPPV was assessed by receiver operating characteristic (ROC) curves. It revealed that the DHI 5-item questionnaire had good internal consistency (Cronbach's coefficient α = 0.72). Area under the curve of total DHI, 5-item and 2-item scores for discriminating BPPV from those without was 0.678 (95 % CI 0.578-0.778), 0.873(95 % CI 0.807-0.940) and 0.895(95 % CI 0.836-0.953), respectively. It revealed 74.5 % sensitivity and 88.7 % specificity in separating BPPV and those without, with a cutoff value of 12 in the 5-item questionnaire. The corresponding rate of sensitivity and specificity was 78.2 and 88.7 %, respectively, with a cutoff value of 6 in 2-item questionnaire. The present study indicated that both 5-item and 2-item questionnaires in the Chinese version of DHI may be more valid than DHI total score for screening objective BPPV and merit further application in clinical practice in China.

SB-431542, a specific inhibitor of the TGF-ß type I receptor inhibits hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts.

The vascular remodeling process plays an important role in the pathology of hypoxia-induced pulmonary hypertension, and it includes cell proliferation, cell motility, cell synthesis and collagen coagulation. Due to their proliferation and synthesis ability, the adventitial fibroblasts are thought to be critical in the vascular remodeling process initiated in response to hypoxia. However, the factors driving hypoxia-induced fibroblast proliferation and synthesis have yet to be elucidated, and the treatment regimens to treat hypoxia remain ineffective. As forthis study, its purpose was to examine the effects exerted by SB-431542, a small-molecule antagonist of transforming growth factor-ß-receptor, on the proliferation, synthesis and collagen coagulation in cultured adventitial fibroblasts. Another aim of this study was to assess the inhibitory ability of SB-431542 on pulmonary vascular remodeling in chronic hypoxia in vivo.The cell morphology and proliferation of cultured adventitial fibroblasts was assessed by laser confocal microscopy and the MTT assay, respectively. Additionally, collagen synthesis was determined by hydroxyproline chromatography, while the expression of cytokines in adventitial fibroblasts and lung tissues was evaluated by immunohistochemical and reverse transcription PCR analyses. The results indicated that the exposure of cultured fibroblasts to 1% oxygen led to the up regulation of cell proliferation, cell synthesis. In addition, increased expression of cytokines and collagen was detected in vivo in the pulmonary artery adventitia of rats exposed to chronic hypoxia. Conversely, SB-431542 inhibited fibroblast proliferation and synthesis in the process of hypoxia-induced pulmonary hypertension (P < 0.01). Thus, the results suggested that by reducing cell proliferation, cell synthesis of vascular adventitia, small molecule inhibitors of the TGF-ß1 receptors may offer a novel therapy for pulmonary hypertension.

Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29.

The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function.

Early fibrinogen degradation coagulopathy: a predictive factor of parenchymal hematomas in cerebral rt-PA thrombolysis.

BACKGROUND AND PURPOSE: The purpose of this study was to systematically determine the correlations between the post-thrombolytic changes of hemostasis parameters and the occurrence of early intracerebral hemorrhage (ICH). METHODS: In 72 consecutive patients with cerebral infarcts treated with rt-PA, plasma levels of fibrinogen, plasminogen, alpha2-antiplasmin, factor XIII, fibrin(ogen) degradation products (FDPs) and d-Dimers were measured at baseline, 2 and 24h after thrombolysis. Correlations were studied between the hemostasis events and early (less than 24h) hemorrhagic infarcts (HIs) or parenchymatous hematomas (PH). RESULTS: Of 72 patients, 6 patients (8.3%) had early PHs, 11 (15.3%) had early HIs, and 55 (76.4%) had no bleeding. Early HIs were not linked to any hemostasis parameter at any time. Univariate comparison of patients having early PHs with non-bleeding patients showed hemostasis abnormalities at 2h: high FDP (p=0.01), high Log FDP (p=0.01), low fibrinogen (p=0.01), and low Log fibrinogen (p=0.01). Logistic regression adjusted for age, NIHSS and diabetes confirmed these 2hour predictors: Log FDP (OR: 7.50; CI: 1.26 to 44.61, p=0.03), and Log fibrinogen (OR: 19.32; CI: 1.81 to 205.98, p=0.01). The decrease in fibrinogen less than 2g/L multiplies the odds of early PH by a factor 12.82. CONCLUSION: An early fibrinogen degradation coagulopathy involving an increase of FDP and a massive consumption of circulating fibrinogen is predictive of early parenchymal hematomas, indicating the occurrence of a particularly intense lysis of circulating fibrinogen. These results, if confirmed by future studies, suggest that early assays of fibrinogen and FDP may be useful in predicting the risk of post-thrombolytic intracerebral hematoma.

Post-thrombolysis haemostasis changes after rt-PA treatment in acute cerebral infarct. Correlations with cardioembolic aetiology and outcome.

BACKGROUND: Little is known, in man, in the post-thrombolytic molecular dynamics of haemostasis, particularly the effect of rt-PA on antifibrinolytic components such as alpha2 anti-plasmin and Factor XIII. AIMS AND HYPOTHESIS: The purpose of this study was to systematically determine changes in coagulation and fibrinolytic parameters after thrombolysis with rt-PA during 24h. We also aimed to correlate these parameters with different acute ischemic stroke subtypes and global outcome. METHODS: Eighty consecutive patients with cerebral infarcts treated with rt-PA had their plasma levels of fibrinogen, plasminogen, alpha2-antiplasmin, Factor XIII, fibrin(ogen) degradation products (FDP) and D-Dimers measured at baseline (h0), 2 (h2) and 24h (h24) after initiation of thrombolysis. Correlations between the variations of these components were statistically studied, using the Spearman rank test or the Pearson test. These haemostatic parameters were also compared with cardioembolic and non cardioembolic patients, as well as between poor and favourable outcome patients. RESULTS: Between h0 and h2, a decrease in fibrinogen, plasminogen, alpha2-antiplasmin, and factor XIII was observed, while an increase in FDP and D-Dimers took place. These values returned to the initial levels at h24. At 2h, the decrease in fibrinogen was significantly correlated with that of plasminogen (0.48, p=0.01), alpha2-antiplasmin (0.48, p=0.004), and factor XIII (0.44, p=0.01); the decrease in plasminogen was significantly correlated with those of antifibrinolytic components, factor XIII (0.47, p=0.02) and alpha2-antiplasmin (r=0.77, p<0.001). These variations were independent of NIHSS. Cardioembolic infarcts showed a statistically significant greater h0-h2 decrease in plasminogen (p=0.04) and an h0-h2 increase in FDP (p=0.02). Poor outcome was linked to low plasminogen values at 2 and 24h. CONCLUSIONS: Supposed to be fibrin-specific, rt-PA induces a decrease in circulating fibrinogen, significantly linked to a decrease in plasminogen. A collateral increase in antifibrinolytic agents such as factor XIII and alpha2-antiplasmin is also observed. At 2h, a significant decrease in plasminogen and a significant increase in fibrin(ogen) degradation products (FDP) are observed in cardioembolic infarcts, and appear as early independent predictors of this aetiology. A low plasminogen value at 2h is potentially predictive of poor prognosis at 3months.

Effects of hydroxysafflor yellow A on proliferation and collagen synthesis of rat vascular adventitial fibroblasts induced by angiotensin II.

THE PURPOSE OF THIS STUDY WAS TO: 1) examine the effects of hydroxysafflor yellow A (HSYA) on the proliferation, collagen and cytokine synthesis of vascular adventitial fibroblasts as induced by angiotensin II (Ang II) in normal Sprague-Dawley (SD) rats in vitro, and 2) to assess the effects of HSYA on morphological changes and collagen accumulation of vascular adventitia in spontaneously hypertensive rats (SHR) in vivo. In vitro experiment, vascular adventitial fibroblasts from SD rats were isolated, cultured, and divided into control groups, model groups and HSYA groups. Cell morphology of adventitial fibroblasts was assessed using laser confocal microscopy, while cell proliferation with the MTT assay, and collagen synthesis was determined using hydroxyproline chromatometry. Immunocytochemistry and reverse transcription PCR were used for detecting the expression of TGF-ß1, MMP-1, α-SMA and NF-κB in adventitial fibroblasts. In vivo experiment, vascular adventitia proliferation and collagen synthesis were analyzed using hematoxylin-eosin and Sirius staining. Our results showed that: 1) in vitro experiment of SD rats, HSYA inhibited proliferative activity and collagen synthesis of adventitial fibroblasts as induced by Ang II, and the inhibitory effects of HSYA on the increased expression of MMP-1, TGF-ß1, α-SMA and NF-κB p65 as induced by Ang II were assessed, and 2) in vivo experiment of SHR, histological analysis displayed fewer pathological changes of vascular adventitia in HSYA treatment groups as compared with no HSYA treatment groups, and MMP-1, TGF-ß1, α-SMA and NF-κB p65 expression significantly reduced after HSYA treatment (P < 0.05). Our results revealed that HSYA treatment significantly decreased the amount of cytokines and collagen synthesis in vascular adventitia components. This study provides experimental evidence demonstrating that HSYA has the capacity to decrease vascular adventitia proliferation and hyperplasia during vascular remodeling.

Effects of BMSCs interactions with adventitial fibroblasts in transdifferentiation and ultrastructure processes.

In this study an in vitro model of simulated blood vessel injury was used to study the effects of bone marrow-derived mesenchymal stem cells (BMSCs) morphology and to detect vascular smooth muscle actin (SM α-actin) expression in the presence of adventitial fibroblasts. BMSCs from rats with DAPI-labeled nuclei were co-cultured with adventitial fibroblasts for 7 days, while BMSCs cultured alone served as controls. Cell morphology of BMSCs was assessed by laser confocal microscopy and SM α-actin or calponin expression in BMSCs was detected by immunofluorescence staining. The expression of SM α-actin mRNA was identified using RT-PCR. Cell ultrastructure was assessed by electron microscopy. The results demonstrate that BMSCs with DAPI-labeled nuclei were smaller compared with fibroblasts, and their nuclei emitted a blue fluorescence. Most BMSCs displayed a polygonal shape changing from their original long fusiform shape. BMSCs with blue nuclei and red cytoplasm (SM α-actin positive or calponin positive) were observed, and a substantial number of filaments were present in the cytoplasm as observed under electron microscopy. The number of these cells increased as a function of culture duration. However, SM α-actin expression was weak and calponin expression was not detected in the control group. This study provides important new information on the characterization of artherosclerosis pathogenesis and vascular restenosis after blood vessel injury. Our findings demonstrate that direct interactions with adventitial fibroblasts can induce vascular smooth muscle-like cell differentiation in BMSCs.