Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable – possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock

Alterations in vasodilator-stimulated phosphoprotein (VASP) phosphorylation: associations with asthmatic phenotype, airway inflammation and β(2)-agonist use

BACKGROUND: Vasodilator-stimulated phosphoprotein (VASP) mediates focal adhesion, actin filament binding and polymerization in a variety of cells, thereby inhibiting cell movement. Phosphorylation of VASP via cAMP and cGMP dependent protein kinases releases this "brake" on cell motility. Thus, phosphorylation of VASP may be necessary for epithelial cell repair of damage from allergen-induced inflammation. Two hypotheses were examined: (1) injury from segmental allergen challenge increases VASP phosphorylation in airway epithelium in asthmatic but not nonasthmatic normal subjects, (2) regular in vivo β(2)-agonist use increases VASP phosphorylation in asthmatic epithelium, altering cell adhesion. METHODS: Bronchial epithelium was obtained from asthmatic and non-asthmatic normal subjects before and after segmental allergen challenge, and after regularly inhaled albuterol, in three separate protocols. VASP phosphorylation was examined in Western blots of epithelial samples. DNA was obtained for β(2)-adrenergic receptor haplotype determination. RESULTS: Although VASP phosphorylation increased, it was not significantly greater after allergen challenge in asthmatics or normals. However, VASP phosphorylation in epithelium of nonasthmatic normal subjects was double that observed in asthmatic subjects, both at baseline and after challenge. Regularly inhaled albuterol significantly increased VASP phosphorylation in asthmatic subjects in both unchallenged and antigen challenged lung segment epithelium. There was also a significant increase in epithelial cells in the bronchoalveolar lavage of the unchallenged lung segment after regular inhalation of albuterol but not of placebo. The haplotypes of the β(2)-adrenergic receptor did not appear to associate with increased or decreased phosphorylation of VASP. CONCLUSION: Decreased VASP phosphorylation was observed in epithelial cells of asthmatics compared to nonasthmatic normals, despite response to β-agonist. The decreased phosphorylation does not appear to be associated with a particular β(2)-adrenergic receptor haplotype. The observed decrease in VASP phosphorylation suggests greater inhibition of actin reorganization which is necessary for altering attachment and migration required during epithelial repair

Search for Gravitational Waves from Primordial Black Hole Binary Coalescences in the Galactic Halo

We use data from the second science run of the LIGO gravitational-wave detectors to search for the gravitational waves from primordial black hole (PBH) binary coalescence with component masses in the range 0.2--$1.0 M_\odot$. The analysis requires a signal to be found in the data from both LIGO observatories, according to a set of coincidence criteria. No inspiral signals were found. Assuming a spherical halo with core radius 5 kpc extending to 50 kpc containing non-spinning black holes with masses in the range 0.2--$1.0 M_\odot$, we place an observational upper limit on the rate of PBH coalescence of 63 per year per Milky Way halo (MWH) with 90% confidence.Comment: 7 pages, 4 figures, to be submitted to Phys. Rev.

Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BACKGROUND: Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated "amino acid adenylation domain". Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on

Attachment and coping in psychosis in relation to spiritual figures

Background: Studies have found higher levels of insecure attachment in individuals with schizophrenia. Attachment theory provides a framework necessary for conceptualizing the development of interpersonal functioning. Some aspects of the attachment of the believer to his/her spiritual figure are similar to those between the child and his/her parents. The correspondence hypothesis suggests that early child-parent interactions correspond to a person's relation to a spiritual figure. The compensation hypothesis suggests that an insecure attachment history would lead to a strong religiousness/spirituality as a compensation for the lack of felt security. The aim of this study is to explore attachment models in psychosis vs. healthy controls, the relationships between attachment and psychopathology and the attachment processes related to spiritual figures. Methods: Attachment models were measured in 30 patients with psychosis and 18 controls with the AAI (Adult Attachment interview) in relationship with psychopathology. Beliefs and practices related to a spiritual figure were investigated by qualitative and quantitative analyses. Results: Patients with psychosis showed a high prevalence of insecure avoidant attachment. Spiritual entities functioned like attachment figures in two thirds of cases. Interviews revealed the transformation of internal working models within relation to a spiritual figure: a compensation process was found in 7 of the 32 subjects who showed a significant attachment to a spiritual figure. Conclusions: Attachment theory allows us to highlight one of the underlying dimensions of spiritual coping in patients with psychosis

Comparing the effectiveness of monetary versus moral motives in environmental campaigning

Environmental campaigns often promote energy conservation by appealing to economic (for example, lower electricity bills) rather than biospheric concerns (for example, reduced carbon emissions), assuming that people are primarily motivated by economic self-interest. However, people also care about maintaining a favourable view of themselves (they want to maintain a 'positive self-concept'), and may prefer to see themselves as 'green' rather than 'greedy'. Consequently, people may find economic appeals less attractive than biospheric appeals. Across two studies, participants indicated feeling better about biospheric ('Want to protect the environment? Check your car's tire pressure') than economic ('Want to save money? Check your car's tire pressure') tyre-check appeals. In a field experiment, we found that an economic tyre-check appeal ('Do you care about your finances? Get a free tire check') elicited significantly less compliance than parallel biospheric and neutral appeals. Together, these studies discredit the conventional wisdom that appealing to economic self-interest is the best way to secure behaviour change. At least in some cases, our studies suggest, this strategy is not effective.</p

The Effect of Aliphatic Carboxylic Acids on Olfaction-Based Host-Seeking of the Malaria Mosquito Anopheles gambiae sensu stricto

The role of aliphatic carboxylic acids in host-seeking response of the malaria mosquito Anopheles gambiae sensu stricto was examined both in a dual-choice olfactometer and with indoor traps. A basic attractive blend of ammonia + lactic acid served as internal standard odor. Single carboxylic acids were tested in a tripartite blend with ammonia + lactic acid. Four different airflow stream rates (0.5, 5, 50, and 100 ml/min) carrying the compounds were tested for their effect on trap entry response in the olfactometer. In the olfactometer, propanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, heptanoic acid, octanoic acid, and tetradecanoic acid increased attraction relative to the basic blend. While several carboxylic acids were attractive only at one or two flow rates, tetradecanoic acid was attractive at all flow rates tested. Heptanoic acid was attractive at the lowest flow rate (0.5 ml/min), but repellent at 5 and 50 ml/min. Mixing the air stream laden with these 7 carboxylic acids together with the headspace of the basic blend increased attraction in two quantitative compositions. Subtraction of single acids from the most attractive blend revealed that 3-methylbutanoic acid had a negative effect on trap entry response. In the absence of tetradecanoic acid, the blend was repellent. In assays with MM-X traps, both a blend of 7 carboxylic acids + ammonia + lactic acid (all applied from low density polyethylene-sachets) and a simple blend of ammonia + lactic acid + tetradecanoic acid were attractive. The results show that carboxylic acids play an essential role in the host-seeking behavior of An. gambiae, and that the contribution to blend attractiveness depends on the specific compound studied

CD4(+)CD25(+)FOXP3(+) Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy

Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodeling. Recently microglial cells have been shown to be responsible for a portion of synaptic remodeling, but the remaining mechanisms remain mysterious. Here we report a new role for astrocytes in actively engulfing CNS synapses. This process helps to mediate synapse elimination, requires the Megf10 and Mertk phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to normally refine their retinogeniculate connections and retain excess functional synapses. Lastly, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify Megf10 and Mertk as critical players in the synapse remodeling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes