Plasminogen activator, tissue type (PLAT) and its inhibitor serpin family E member 1 (SERPINE1) cooperatively regulate PLAT activity in various reproductive processes. However, it is unknown whether this includes bovine oocyte maturation. We addressed this question in the present study by evaluating PLAT and SERPINE1 protein localization in immature cumulus-oocyte complexes (COCs), as well as PLAT mRNA and protein expression in cultured COCs after 0, 8, 16, and 24 h of in vitro maturation (IVM). We also examined the effects of PLAT and SERPINE1 on germinal vesicle breakdown (GVBD) and oocyte cyclic 3' 5' adenosine monophosphate (cAMP) levels, cumulus expansion index, and expansion-related gene expression in oocytes derived from bovine COCs cultured for 4, 8, and 12 h and in COCs cultured for 16 h. Both PLAT and SERPINE1 localized in cumulus cells but only the latter was detected in oocytes. PLAT and SERPINE1 transcript levels increased during IVM; however, from 8 to 16 h, the levels of PLAT remained stable whereas those of SERPINE1 increased, resulting in a decline in PLAT concentration. Additionally, PLAT delayed GVBD, increased oocyte cAMP levels, and blocked cumulus expansion and associated gene expression, which was reversed by SERPINE1 supplemented. Thus, PLAT delays bovine oocyte GVBD by enhancing oocyte cAMP levels during the first 8 h of IVM; suppression of PLAT activity via accumulation of SERPINE1 in COCs results in cumulus expansion from 8 to 16 h of IVM. These findings provide novel insights into the molecular mechanisms underlying in vitro bovine oocyte maturation.

This study attempts to identify coding risk variants in genes previously implicated in Alzheimer's disease (AD) pathways, through whole-exome sequencing of subjects (N = 17) with AD, with a positive family history of dementia (familial AD). We attempted to evaluate the mutation burden in genes encoding amyloid precursor protein metabolism and previously linked to risk of dementias. Novel variants were identified in genes involved in amyloid precursor protein metabolism such as PSEN1 (chr 14:73653575, W161C, tgg>> tgT), PLAT (chr 8:42039530,G272R), and SORL1 (chr11:121414373,G601D). The mutation burden assessment of dementia-related genes for all 17 cases revealed 45 variants, which were either shared across subjects, or were present in just the 1 patient. The study shows that the clinical characteristics, and genetic correlates, obtained in this sample are broadly comparable to the other studies that have investigated familial forms of AD. Our study identifies rare deleterious genetic variations, in the coding region of genes involved in amyloid signaling, and other dementia-associated pathways.

Paxillin (PXN), a key component of the focal adhesion complex, has been associated with cancer progression, but the underlying mechanisms are poorly understood. The purpose of this study was to elucidate mechanisms by which PXN affects cancer growth and progression, which we addressed using cancer patient data, cell lines, and orthotopic mouse models. We demonstrated a previously unrecognized mechanism whereby nuclear PXN enhances angiogenesis by transcriptionally regulating SRC expression. SRC, in turn, increases PLAT expression through NF-ĸB activation; PLAT promotes angiogenesis via LRP1 in endothelial cells. PXN silencing in ovarian cancer mouse models reduced angiogenesis, tumor growth, and metastasis. These findings provide a new understanding of the role of PXN in regulating tumor angiogenesis and growth.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

The endoplasmic reticulum stress is an important factor of tumor growth and is induced in cancer cells. We have studied the effect of ERN1 knockdown as well as hypoxia on the expression of genes encoding factors, which control cell proliferation, in U87 glioma cells. It was shown that the complete blockade of ERN1 enzyme function leads to an increase of the PLAT (tissue plasminogen activator), CCN2 (CCN family member 2), and ITGB1 (integrin β-1) as well as to a decrease ofPLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), and SLURP1 (secreted LY6/PLAUR domain containing 1) mRNA expressions. Moreover, we have shown that hypoxia does not affect the expression level of ITGB1 mRNA, but increases that of CCN2, PLAUR, SLURP1, and PLAT mRNA and decreases the expression level of only PLAU mRNA in control glioma cells. At the same time, in ERN1 knockdown glioma cells the expression level of PLAU PLAUR, and SLURP1 mRNA is decreased under hypoxia, but PLAT and ITGB1 mRNA expression levels are increased under these experimental conditions. Thus, results of this study have shown that the expression level of all studied genes is affected by ERN1 knockdown as well as by hypoxia and that the effect of hypoxia mostly depends on ERN1 signaling enzyme function.

OBJECTIVE

To assess the effect of mehanical ventilation (MV) guided by transpulmonary pressure (Ptp) on respiratory mechanics and gas exchange in severe acute pancreatitis patient with intraabdominal hypertension.

METHODS

Twelve severe acute pancreatitis patient with intraabdominal hypertension and acute respiratory distress syndrome(ARDS) underwent mechanical ventilation were involved from Jan to Dec 2013. PEEP levels were set to achieve a Ptp of 0 to 10 cm of water at end expiration. We also limited tidal volume to keep Ptp at less than 25 cm of water at end inspiration. Respiratory mechanics and gas-exchange were measured.

RESULTS

Plat pressure (Pplat) increased and the compliance of chest wall (Ccw) decreased when intraabdominal pressure (IAP) increased. Pplat correlated with IAP positively (r2=0.741 9, P<0.05) and Ccw correlated with IAP negtively (r2=0.722 2, P<0.05), respectively.There were not corrletions between IAP and end-expiratory Ptp (Ptp-e) and end-inspiratory Ptp (Ptp-i) (P>0.05). Compared with baseline, after guiding MV with Ptp, the Level of PEEP (14.6±4.2) cmH2O vs (8.3±2.0) cmH2O, and Ptp-e (1.5±0.5) cmH2O vs (-2.3±1.4) cmH2O increased (P<0.05) and Ptp-i did not increase significantly (P>0.05). Ptp-e correlated with PEEP (r2=0.549, P<0.05) and end-expiratory esophageal pressure (Pes-e) (r2=0.260, P<0.05). Ptp-i correlated with Pplat (r2=0.523, P<0.05) and end-inspiratory esophageal pressure (Pes-i) (r2=0.231, P<0.05), but did not correlate with Tidal volume(VT) (r2=0.052 4, P>0.05). Compared with baseline, lung compliance (CL) (48.1±10.3) cmH2O vs (25.7±6.4) cmH2O and oxygenation index (PaO2/FiO2) (235±48) mmHg vs (160±35) mmHg improved obviously (P<0.05), dead space fraction (VD/VT) (0.48±0.07) vs (0.59±0.06) decreased (P<0.05), but Ccw and respiratory compliance(Cr) didn't improve (P>0.05).

CONCLUSIONS

Transpulmonary pressure-directed mechanical ventilation in ARDS secondary to severe acute pancreatitis patient with intraabdominal hypertension could not only recruit the collapsed alveoli, improve lung compliance, increase oxygenation index and decrease dead space ventilation but also monitor lung stress to avoid alveoli overinflation, which might be lung protective.

OBJECTIVE

To determine the one-year incidence of nonfatal farm injuries among women actively engaged in farm work; to determine the distribution of these injuries by external cause, severity, and other characteristics; and to determine the potential risk factors for injuries among farm women.

METHODS

A population-based study of the occurrence of agricultural injuries during the previous year was conducted in a stratified, random sample of 1096 actively working farm women in Texas and Louisiana. Sampling pools were generated from county plat listings in Texas and maintained mailing lists in Louisiana; counties were selected based on geographical and agricultural diversity. Eligible women were interviewed by phone about injury occurrences and patterns.

RESULTS

The cumulative one-year incidence of farm injuries for women in this area was 5% (95% CI = 3.7-6.3), based on the number of farm women injured. Lower extremities were the most frequently injured body parts. The leading external causes were contact with foreign object/substance, falls, and overdoing/lifting/hauling. Most injuries occurred in the summer or spring. Factors predictive of increased injury risk in adjusted logistic regression included working on large-animal farms, more time spent in farm work, persistent back pain or weakness during the previous 12 months, driving a tractor, and hauling farm goods to market. Most women consulted physicians as a result of their injuries.

CONCLUSIONS

Physicians are in a unique position to institute interventions to prevent risky behaviors.

Background: A total of 68% of pre-school children with cleft palate have speech problems requiring speech therapy. There is a lack of access to regular targeted therapy. Parent training leads to positive outcomes in early communication skills in cleft palate and non-cleft speech disorders. Connected health has been used to address inadequate access to therapy, providing intervention to those who would not otherwise receive therapy.

Aims: To evaluate the speech, activity and participation outcomes of Parent Led, Therapist Supervised, Articulation Therapy (PLAT) compared with routine speech therapy intervention in parent-child dyads.

Methods & procedures: A total of 44 children, aged 2.9-7.5 years, were included in a two-centre, two-phase randomized controlled trial. Informed consent and assent were obtained. Participants and speech and language therapists (SLTs) were unblinded to the groups. Parents, in the parent-trained group (n = 23), attended 2 days' training, received a detailed speech therapy programme, and undertook intervention over 12 weeks supported by the cleft specialist SLT using FaceTime and one face-to-face session. In the control arm (n = 21), parent-child dyads received six therapy sessions over 12 weeks with a research SLT, comparable with usual care. Speech recordings were undertaken pre- and post-intervention. Percent consonant correct (PCC) was analysed by external SLTs blinded to the time and group. Activity and participation were measured using the Intelligibility in Context Scale (ICS) and Focus on Outcomes for Children Under Six (FOCUS) questionnaire.

Outcomes & results: There was no evidence of an interaction between Time and Group or an overall statistical difference between groups for PCC scores. There was a statistically significant difference over time for both groups (words: p < 0.002; confidence interval (CI) = 9.38-16.27; d = 0.57; sentences: p < 0.002; CI = 16.04-25.97; d = 0.23). Effect sizes were medium for words and small for sentences. For intelligibility and participation, there was no evidence of an interaction between Time and Group or an overall statistical difference between groups. A statistically significant difference over time was found for intelligibility (F = 29.97, d.f. = 1, 42, p < 0.001, 95 % CI = 1.45-3.15 d = 0.46) and for participation (F = 14.19, d.f. = 1, 41, p < 0.001 95% CI = 7.63-25.03; d = 0.36) with FOCUS results indicating clinically meaningful (parent-led group) and significant (control group) change in participation.

Conclusions & implications: PLAT can be as effective as routine care in changing speech, activity and participation outcomes for children with cleft palate, when supported by a specialist cleft SLT using connected health. What this paper adds What is already known on this subject Over 50% of children with cleft palate require speech therapy. However, there is a lack of timely, accessible speech therapy services in the UK and Ireland. Previous studies have shown that parents can deliver therapy effectively, and that connected health can support the delivery of speech therapy. This study aims to provide evidence that parent-led therapy with the supervision of a specialist cleft therapist using FaceTime is effective. What this paper adds to existing knowledge This randomized controlled trial indicates that parents can be trained to deliver therapy for children with cleft palate speech disorders, under the supervision of an SLT. This approach results in improved speech, activity and participation outcomes similar to routine care. What are the potential or actual clinical implications of this work? This study indicates that both parent-led articulation therapy and routine care showed meaningful gains in speech, activity and participation, and that parent-led articulation therapy when supported by a cleft SLT using connected health could be an additional service delivery model for children with cleft palate speech disorders.

Keywords: cleft palate; intervention; speech disorders; trial.

This report describes a simple and efficient system for construction of recombinant pseudorabies (Aujeszky's disease) virus (PrV) which is based on the use of a unique restriction site inserted into the viral genome. This system enables the recovery of genetically modified viruses without screening or selection for a specific phenotype, since practically all mature viral particles obtained carry the foreign sequences. To demonstrate, we introduced the tumour suppressor protein-53 (p53) gene into two different intergenic locations of PrV: the ribonucleotide reductase (rr) gene and the promoter of a putative latency gene (PLAT), located at the inverted repeat (IR) region of the viral genome. As a first step, we engineered a unique EcoRI recognition site into the rr gene or into both copies of PLAT with the help of marker transfer using the bacterial lacZ gene. Then, in both cases viral DNAs were cut with the restriction endonuclease EcoRI followed by treatment with calf intestinal phosphatase and used for cotransfection into porcine kidney cells with a plasmid containing the p53 gene flanked by viral DNAs homologous to the target region. As a result of this process, in most of the experiments, we obtained recombinant viruses without the background of parental viruses. Here we show that this method can be used for directional insertion of exogenous sequences into either the unique or the IR region of the PrV chromosome. In principle, this system should be applicable to the construction of recombinant derivatives of any viruses having infectious DNA.

OBJECTIVE

High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy.

METHODS

DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries.

RESULTS

Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction.

CONCLUSIONS

Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Increased concern about potential losses of phosphorus (P) from agricultural fields receiving animal waste has resulted in the implementation of new state and federal regulations related to nutrient management. In response to strengthened nutrient management standards that require consideration of P, North Carolina has developed a site-specific P indexing system called the Phosphorus Loss Assessment Tool (PLAT) to predict relative amounts of potential P loss from agricultural fields. The purpose of this study was to apply the PLAT index on farms throughout North Carolina in an attempt to predict the percentage and types of farms that will be forced to change management practices due to implementation of new regulations. Sites from all 100 counties were sampled, with the number of samples taken from each county depending on the proportion of the state's agricultural land that occurs in that county. Results showed that approximately 8% of producers in the state will be required to apply animal waste or inorganic fertilizer on a P rather than nitrogen basis, with the percentage increasing for farmers who apply animal waste (approximately 27%). The PLAT index predicted the greatest amounts of P loss from sites in the Coastal Plain region of North Carolina and from sites receiving poultry waste. Loss of dissolved P through surface runoff tended to be greater than other loss pathways and presents an area of concern as no best management practices (BMPs) currently exist for the reduction of in-field dissolved P. The PLAT index predicted the areas in the state that are known to be disproportionately vulnerable to P loss due to histories of high P applications, high densities of animal units, or soil type and landscapes that are most susceptible to P loss.

Folates and tetrahydrofolates inhibit beef liver glutamate dehydrogenase (EC 1.4.1.2). Double reciprocal plats indicate a competitive inhibition for alpha-ketoglutarate-glutamate by folic acid and methotrexate and a complex or mixed type for NAD-NADH site. Pteroic acid is not inhibitory at the concentrations studied. The addition of up to four gamma-linked glutamyl residues to folic and tetrahydrofolic acids increases the inhibition. Further chain elongation of the gamma-peptide had no effect on the inhibitory activity. The p-aminobenzoate poly-gamma-glutamates were less inhibitory than the corresponding folyl polyglutamates.

5-Lipoxygenase (5-LOX) is the key player of pro-inflammatory leukotriene biosynthesis. Its regulatory or so-called PLAT (polycystin-1, lipoxygenase, α-toxin) domain binds allosteric modulators like calcium, membranes, coactosin-like protein and Dicer, thereby influencing 5-LOX activity at the nuclear membrane by mediating translocation. The PLAT domain may also regulate cytosolic 5-LOX activity and possibly influence microRNA metabolism. Hence, it has also evolved as a promising target for anti-inflammatory therapy. Research focusing on this substructure of 5-LOX requires an assay system based on the isolated domain. However, we found that the isolated PLAT domain was highly prone to aggregation and therefore unsuitable for interaction studies. Substitution of the single, membrane-binding tryptophan 75 with glycine reduced aggregation and substantially increased its thermal stability. Calcium interaction of the single mutant was confirmed by differential scanning fluorimetry. Moreover, crosslinking experiments demonstrated the ability of the isolated PLAT domain to bind Dicer C-terminus whereas the interaction with coactosin-like protein required the interplay of the catalytic and the PLAT domain.

Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.

Polycystin-1 is a large transmembrane protein mutated in the common genetic disorder autosomal dominant polycystic kidney disease. One of the predicted intracellular domains of polycystin-1 is PLAT (Polycystin-1, Lipoxygenase and Alpha Toxin), which consists of 116 amino acids and is anchored to the membrane by linkers at both ends. It is predicted to have a large number of hydrophobic residues on the surface. Assignment of the NMR spectrum was hampered by considerable line broadening, and hence a programme of site-directed mutagenesis and searching for suitable solution conditions was undertaken. The optimum construct required fusion of the GB1 domain at the N-terminus and a His tag at the C-terminus, and proved to have several additional amino acids at both ends beyond the canonical domain boundaries, as well as mutation of W3128 to alanine. Optimum solubility required 500 mM sodium chloride, and usable spectra could only be obtained by perdeuteration. Backbone assignment was made using standard triple resonance spectra and is 88 % complete. The chemical shifts obtained suggest that a loop consisting of residues 3223-3228 is mobile in solution, and that the protein is similar in structure to a prediction produced by Swiss-Model based on the structure of a homologous protein.

Fibrinolysis is initiated by tissue-type plasminogen activator (tPA) and inhibited by plasminogen activator inhibitor 1 (PAI-1). In obese humans, plasma PAI-1 and tPA proteins are increased, but PAI-1 dominates, leading to reduced fibrinolysis and thrombosis. To understand tPA-PAI-1 regulation in obesity, we focused on hepatocytes, a functionally important source of tPA and PAI-1 that sense obesity-induced metabolic stress. We showed that obese mice, like humans, had reduced fibrinolysis and increased plasma PAI-1 and tPA, due largely to their increased hepatocyte expression. A decrease in the PAI-1 (SERPINE1) gene corepressor Rev-Erbα increased PAI-1, which then increased the tPA gene PLAT via a PAI-1/LRP1/PKA/p-CREB1 pathway. This pathway was partially counterbalanced by increased DACH1, a PLAT-negative regulator. We focused on the PAI-1/PLAT pathway, which mitigates the reduction in fibrinolysis in obesity. Thus, silencing hepatocyte PAI-1, CREB1, or tPA in obese mice lowered plasma tPA and further impaired fibrinolysis. The PAI-1/PLAT pathway was present in primary human hepatocytes, and associations among PAI-1, tPA, and PLAT in livers from obese and lean humans were consistent with these findings. Knowledge of PAI-1 and tPA regulation in hepatocytes in obesity may suggest therapeutic strategies for improving fibrinolysis and lowering the risk of thrombosis in this setting.

Keywords: Coagulation; Hematology; Metabolism; Obesity.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvi-ronment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable plat-form to investigate membrane proteins by a broad range of surface-sensitive techniques such as Neutron Reflectometry (NR), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR), Atomic Force Microscopy (AFM) and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challeng-ing. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to medi-ate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and Tissue Factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface and the peptide mol-ecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of mem-brane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile and we show its general applicability by characterizing with the above mentioned surface sensitive techniques two dif-ferent membrane proteins, which were efficiently loaded in the supported bilayers with ~0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins, and for preparation of suitable platforms for drug testing or biosen-sor development.

Realizing superhydrophobicity, high transparency on polydimethylsiloxane (PDMS) surface enlarges its application fields. We applied a femtosecond laser to fabricate well-designed structures combining microgrooves with microholes array on mirror finished stainless steel to form a template. Then liquid PDMS was charged for the duplicating process to introduce a particular structure composed of a microwalls array with a certain distance between each other and a microprotrusion positioned at the center of a plate surrounded by microwalls. The parameters such as the side length of microwalls and the height of a microcone were optimized to achieve required superhydrophobicity at the same time as high-transparency properties. The PDMS surfaces show superhydrophobicity with a static contact angle of up to 154.5 ± 1.7° and sliding angle lower to 6 ± 0.5°, also with a transparency over 91%, a loss less than 1% compared with plat PDMS by the measured light wavelength in the visible light scale. The friction robust over 100 cycles by sandpaper, strong light stability by 8 times density treatment, and thermal stability up to 325 °C of superhydrophobic PDMS surface was investigated. We report here a convenient and efficient duplicating method, being capable to form a transparent PDMS surface with superhydrophobicity in mass production, which shows extensive application potentials.

With the development in DNA self-assembly technology, DNA origami nanostructures have been widely applied in biomedical research. Solid-state nanopores represent an emerging single-molecule sensing platform for studying nanostructures with arbitrary dimensions and physical characteristics, including DNA origami. Here, we employed relatively narrow silicon nitride nanopores to detect the deformation and translocation of DNA origami nanoplates with dimensions of approximately 60×54 nm. We performed translocation experiments using three nanopore diameters that are all smaller than the plat dimensions. Analysis of current blockade signals and the representative events reveals three types of translocation orientations for the nanoplates. Furthermore, by studying the electrical signal characteristics (current change, dwell time) for the different diameter pores, we obtained information about the translocation behaviors for the DNA nanoplates through different constrictions. Our investigation provides an approach to analyze the deformation and translocation of DNA origami structures.

"Poly-plat", SSP, and SAP are second generation analogs of cisplatin (CDDP) with higher efficacy and potency. In order to understand the mechanism of action of these compounds, isolated murine peritoneal macrophages were treated with "poly-plat", SSP, or SAP (5 micrograms/ml) for 2 h. Treated macrophages demonstrated an increase in the number of lysosomes, but only "poly-plat" and SSP treated macrophages were stimulated to form the cytoplasmic extensions so very characteristic of cisplatin after 2 h and 24 h post-treatment. SAP showed cytoplasmic extensions only after 24 h post-treatment, and demonstrated a back to the normal discoid form when viewed at 24 h post-treatment. When drug treated macrophages were co-incubated with S180 tumor cells, cytoplasmic extensions of the macrophages developed contacts, and cytoplasmic continuity with the tumor cells, and a subsequent transfer of lysosomes from macrophage to tumor cell was observed after only 2 h of co-incubation. After 24 h of co-incubation, lysis of S180 cells was achieved. Analysis of the tissue culture supernatants collected from "poly-plat", SSP, and SAP treated macrophages demonstrated the enhanced activity of interleukin-1 alpha of over 400 pg/ml after 2 h post-treatment, compared to only 300 pg/ml with cisplatin 24 h post-treatment. However, only SSP demonstrated an increase in TNF-alpha activity (2000 pg/ml) after 2 h post-treatment, which is comparable to that of cisplatin. Based on our observations we propose that "poly-plat", SSP, and SAP activate various cytolytic factors of the immune system better, than cisplatin.

15-Lipoxygenase-2 protein has been reported to play an important role in normal development of prostate, lung, skin, and cornea tissues. It behaves as a suppressor of prostate cancer development by restricting cell cycle progression and implicating a possible protective role against tumor formation. On the basis of the above report, we selected 15-LOX-2 protein to study the structural classification and functional relationship with associated protein network at computational level. Sequence alignment and protein functional study shows that it contains a highly conserved LOX motif. PLAT domain with PF01477 and LH2 domain with PF00305 were successfully observed. Arachidonate 5-lipoxygenase (PDB ID: 3O8Y) was selected as a template with 42% identity. 3D structure was successfully predicted and verified. Qualitative analysis suggests that the predicted model was reliable and stable with best quality. Quantitative study shows that the model contained expected volume and area with best resolution. Predicted and best evaluated model has been successfully deposited to PMDB database with PMDB ID PM0078035. Active site identification revealed GLU(369), ALA(370), LEU(371), THR(372), HIS(373), LEU(374), HIS(376), SER(377), HIS(378), THR(385), LEU(389), HIS(394), PHE(399), LYS(400), LEU(401), ILE(403) and PRO(404) residues may play a major role during protein-protein, protein-drug and protein-cofactor interactions. STRING database result indicated that IL (4), GPX (2 and 4), PPARG, PTGS (1 and 2), CYP (2J2, 2C8, 4A11 and 2B6), PLA (2G2A, 2G4A, 2G1B and 2G6) and A LOX (5, 15, 12 and 12B) members from their respective gene families have network based functional association with 15-LOX-2.

MicroRNAs(miRNAs)are small non-coding RNAs that function in diverse biological processes and are approximately 20-22 nucleotide RNAs that regulate the expression of target genes, mainly at the post-transcriptional level. A number of studies report that miRNAs are involved in homeostatic maintenance such as cell cycle regulation, cell division and apoptosis, and that aberrant expression of miRNAs is often detected in various types of diseases, including cancer. In cancer biology, miRNAs play functional roles in tumor seeding, drug sensitivity, and metastasis. MiRNAs are also secreted through the small vesicles called exosomes, which are endosome-derived vesicles from various cell types including immune and tumor cells. In addition to cellular miRNAs, secreted miRNAs also play important roles in cancer development and metastasis. Therefore, secreted miRNAs in body fluids have been investigated as a promising biomarkers and therapeutic targets for the treatment of cancer patients. In this review, we introduce the current knowledge of miRNA functions in cancer development and discuss the clinical applications of se-miRNAs, eg, as diagnostic markers and therapeutic targets.

Heterogeneity in drug sensitivity must, in part, account for the relative lack of success with single agent chemotherapy for glioblastoma multiforme (GBM). In order to develop in vitro model systems to investigate this, clones derived from the VM spontaneous murine astrocytoma have been characterised with regard to drug sensitivity. Six clonal cell lines have been tested for sensitivity to a panel of cytotoxic drugs using an intermediate duration 35S-methionine uptake assay. These lines have previously been extensively characterised with regard to morphological, antigenic, kinetic, tumourigenic potential in syngeneic animals and chromosomal properties and display considerable heterogeneity. The present study indicates that heterogeneity extends to sensitivity to all classes of cytotoxic drugs. The greatest difference in sensitivity between the clones was seen in response to cell cycle-specific drugs like the Vinca alkaloids (14-fold and 20-fold for vincristine (VCR) and vindesine (VIND) respectively), while the nitrosoureas, CCNU and BCNU displayed a smaller fold difference in sensitivity (4.3 and 3.6-fold difference respectively). All the clones were considerably more resistant to the adriamycin (ADM), cis-platinum (C-PLAT) and the Vinca alkaloids than the parental cell line although the difference in sensitivity between the clones and parental cell line were less marked for the nitrosoureas and procarbazine (PCB). It has also been possible to examine the relationship between drug sensitivity and the phenotypic and genotypic properties of these clonal cell lines. There is a relationship between chromosome number and sensitivity of a wide variety of cytotoxic drugs including the nitrosoureas, Vinca alkaloids, PCB, C-PLAT, BLEO but not ADR or 5-FU. Clones with small numbers of chromosomes were more resistant than clones with gross polyploidy. Similarly, sensitivity to Vinca alkaloids and ADM, but not other classes of drugs, was greatest in cells with numerous cytoplasmic processes and which did not express large amounts of cell surface fibronectin. Preliminary experiments have been conducted on reconstituting clonal mixtures of cells with different sensitivity to Vinca alkaloids and results from these studies indicate that the drug resistance phenotype is dominant, with clonal mixtures of sensitive and resistant cell adopting the sensitivity of the more resistant partner. These cell lines should prove to be useful models for examining the cell biological basis of drug resistance in glioma and may lead to the identification and exploitation of novel cellular targets in new therapies for GBM.