Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems. © 2010 Elsevier Inc.

American Alligator Digestion Rate of Blue Crabs and Its Implications for Stomach Contents Analysis

Stomach contents analysis (SCA) provides a snap-shot observation of a consumer's diet. Interpretation of SCA data can be complicated by many factors, including variation in gastric residence times and digestion rates among prey taxa. Although some SCA methods are reported to efficiently remove all stomach contents, the effectiveness of these techniques has rarely been tested for large irregular shaped prey with hard exoskeletons. We used a controlled feeding trial to estimate gastric residency time and decomposition rate of a large crustacean prey item, the Blue Crab (Callinectes sapidus), which is consumed by American Alligators (Alligator mississippiensis), an abundant apex predator in coastal habitats of the southeastern United States. The decomposition rate of C. sapidus in the stomachs of A. mississippiensis followed a predictable pattern, and some crab pieces remained in stomachs for at least 14 days. We also found that certain portions of C. sapidus were prone to becoming caught within the stomach or esophagus, meaning not all crab parts are consistently recovered using gastric lavage techniques. However, because the state of decomposition of crabs was predictable, it is possible to estimate time since consumption for crabs recovered from wild alligators. This information, coupled with a detailed understanding of crab distributions and alligator movement tactics could help elucidate patterns of cross-ecosystem foraging by the American Alligator in coastal habitats.

Antibacterial Activity of Extracellular Products from Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that can be found in freshwater and marine environments as well as in soil. These organisms produce a variety of different biologically active compounds exhibiting anti-bacterial, anti-fungal and anti-cancer properties among others. In this study, cyanobacterial isolates were screened for their ability to produce extracellular antibacterial products. Cyanobacteria were isolated from fresh water and soil samples collected in the Pembroke Pines, FL area. Twenty- seven strains of cyanobacteria were isolated belonging to the following genera: Limnothrix, Nostoc, Fischerella, Anabaena, Pseudoanabaena, Lyngbya, Leptolyngbya, Tychonema, and Calothrix. Individual strains were grown in liquid culture in laboratory conditions. Following 14-day cultivation, the culture liquid was filtered and tested for activity against the following bacteria: Escherichia coli, Bacillus megatarium, Staphylococcus aureus, and Micrococcus luteus. Among all genera of cyanobacterial strains tested, Fischerella exhibited the greatest inhibitory activity. An attempt was made to isolate the active compound from the culture liquid of the active strains. Lipophilic extracts from culture liquid were obtained from three selected Fischerella strains. The extracts proved to have varying levels of activity against the tested bacteria. Inhibitory activity from all three Fischerella strains was detected against B. megatarium and M luteus. The only strain that was active against S. aureus was Fischerella sp. 114-12 while none of the extracts showed activity against E. coli. This kind of screening has potential pharmaceutical and agricultural benefits, including possible discovery of novel antibiotics.

Vesicoureteral reflux and the extracellular matrix connection.

Primary vesicoureteral reflux (VUR) is a common pediatric condition due to a developmental defect in the ureterovesical junction. The prevalence of VUR among individuals with connective tissue disorders, as well as the importance of the ureter and bladder wall musculature for the anti-reflux mechanism, suggest that defects in the extracellular matrix (ECM) within the ureterovesical junction may result in VUR. This review will discuss the function of the smooth muscle and its supporting ECM microenvironment with respect to VUR, and explore the association of VUR with mutations in ECM-related genes.

The extracellular matrix contributes to mechanotransduction in uterine fibroids.

The role of the extracellular matrix (ECM) and mechanotransduction as an important signaling factor in the human uterus is just beginning to be appreciated. The ECM is not only the substance that surrounds cells, but ECM stiffness will either compress cells or stretch them resulting in signals converted into chemical changes within the cell, depending on the amount of collagen, cross-linking, and hydration, as well as other ECM components. In this review we present evidence that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (1) resistance to apoptosis leading to the persistence of cells and (2) secretion of collagen and other components of the ECM such a proteoglycans by those cells leading to abundant disposition of highly cross-linked, disoriented, and often widely dispersed collagen fibrils. Fibrosis affects cell growth by mechanotransduction, the dynamic signaling system whereby mechanical forces initiate chemical signaling in cells. Data indicate that the structurally disordered and abnormally formed ECM of uterine fibroids contributes to fibroid formation and growth. An appreciation of the critical role of ECM stiffness to fibroid growth may lead to new strategies for treatment of this common disease.

Seawater carbonate chemistry and conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: Acid-base status, trace elements and delta11B, 2012

Mytilus edulis were cultured for 3 months under six different seawater pCO2 levels ranging from 380 to 4000 µatm. Specimen were taken from Kiel Fjord (Western Baltic Sea, Germany) which is a habitat with high and variable seawater pCO2 and related shifts in carbonate system speciation (e.g., low pH and low CaCO3 saturation state). Hemolymph (HL) and extrapallial fluid (EPF) samples were analyzed for pH and total dissolved inorganic carbon (CT) to calculate pCO2 and [HCO3]. A second experiment was conducted for 2 months with three different pCO2 levels (380, 1400 and 4000 µatm). Boron isotopes (delta11B) were investigated by LA-MC-ICP-MS (Laser Ablation-Multicollector-Inductively Coupled Plasma-Mass Spectrometry) in shell portions precipitated during experimental treatment time. Additionally, elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF of specimen from the second experiment were measured via ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Extracellular pH was not significantly different in HL and EPF but systematically lower than ambient water pH. This is due to high extracellular pCO2 values, a prerequisite for metabolic CO2 excretion. No accumulation of extracellular [HCO3] was measured. Elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF increased slightly with pH which is in accordance with increasing growth and calcification rates at higher seawater pH values. Boron isotope ratios were highly variable between different individuals but also within single shells. This corresponds to a high individual variability in fluid B/Ca ratios and may be due to high boron concentrations in the organic parts of the shell. The mean delta11B value shows no trend with pH but appears to represent internal pH (EPF) rather than ambient water pH.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Modest elevation in BNP in asymptomatic hypertensive patients reflects sub-clinical cardiac remodeling, inflammation and extracellular matrix changes

In asymptomatic subjects B-type natriuretic peptide (BNP) is associated with adverse cardiovascular outcomes even at levels well below contemporary thresholds used for the diagnosis of heart failure. The mechanisms behind these observations are unclear. We examined the hypothesis that in an asymptomatic hypertensive population BNP would be associated with sub-clinical evidence of cardiac remodeling, inflammation and extracellular matrix (ECM) alterations. We performed transthoracic echocardiography and sampled coronary sinus (CS) and peripheral serum from patients with low (n = 14) and high BNP (n = 27). Peripheral BNP was closely associated with CS levels (r = 0.92, p<0.001). CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP (r = 0.44, p = 0.008), CITP (r = 0.35, p = 0.03) and PIIINP (r = 0.35, p = 0.001), and with CS levels of inflammatory cytokines including: TNF-α (r = 0.49, p = 0.002), IL-6 (r = 0.35, p = 0.04), and IL-8 (r = 0.54, p<0.001). The high BNP group had greater CS expression of fibro-inflammatory biomarkers including: CITP (3.8±0.7 versus 5.1±1.9, p = 0.007), TNF-α (3.2±0.5 versus 3.7±1.1, p = 003), IL-6 (1.9±1.3 versus 3.4±2.7, p = 0.02) and hsCRP (1.2±1.1 versus 2.4±1.1, p = 0.04), and greater left ventricular mass index (97±20 versus 118±26 g/m(2), p = 0.03) and left atrial volume index (18±2 versus 21±4, p = 0.008). Our data provide insight into the mechanisms behind the observed negative prognostic impact of modest elevations in BNP and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and ECM alterations.

Extracellular vesicle biogenesis in helminths: more than one route to the surface?

The quite recent discovery that parasites release extracellular vesicles (EVs) that can transfer a range of effector molecules to host cells has made us re-think our understanding of the host-parasite interface. In this opinion article we will consider how recent proteomics and transcriptomics studies, together with ultrastructural observations, suggest that more than one mechanism of EV biogenesis can occur in helminths. We propose that distinct EV sub-types have roles in immune-modulation and repair of drug-induced damage, and put forward the case for targeting EV biogenesis pathways to achieve parasite control. In doing so we raise a number of outstanding research questions that must be addressed before this can happen.

Assessment of genotoxicity of aflatoxin M1 and B1 contaminated milks after in vitro human digestion

Introduction - Milk is considered a complete food from the nutritional point of view. Milk can be exposed to various types of contamination, such as mycotoxins. These metabolites are naturally occurring toxic compounds produced by fungi. Several studies on milk samples have reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1), due to the high incidence in samples intended for human consumption, carcinogenicity proven AFB1 and resistance of the contaminants to the process of digestion, making those available for intestinal absorption. Considering these aspects, the objective of this study was to evaluate the genotoxicity of milk samples contaminated by AFB1 and AFM1 before and after the action of lactic acid bacteria using Caco-2 intestinal human cells.

Co-digestion of terrestrial plant biomass with marine macro-algae for biogas production

This paper investigates factors affecting anaerobic degradation of marine macro-algae (or seaweed), when used as a co-substrate with terrestrial plant biomass for the production of biogas. Using Laminaria digitata, a brown marine seaweed species and green peas, results showed that when only 2% of feedstock of a reactor treating the green peas at an organic loading rate (OLR) of 2.67 kg VS.m3.day-1 was replaced with the seaweed, methane production was disrupted, whilst acidogenesis, seemed to be less adversely affected, resulting in excessive volatile acids accumulation. Reactor stability was difficult to achieve thereafter. The experiment was repeated with a lower initial OLR of green peas of 0.70 kg VS.m3.day-1 before the addition of the seaweed. Although similar symptoms as in first trial were observed, process stability was restored through the control of OLR and alkalinity. These measures led to an increase in overall OLR of 1.25 kg VS.m3.day-1 comprising of 35% seaweed. This study has shown that certain seaweed constituents are more inhibitory to the methanogens even at trace concentrations than to the other anaerobic digestion microbial groups. Appropriate adaptation strategy, involving initial low proportion of the seaweed relative to the total OLR, and overall low OLR, is necessary to ensure effective adaptation of the microorganisms to the inhibitory constituents of seaweed. Where there is seasonal availability of seaweed, the results of this study suggest that a fresh adaptation or start-up strategy must be implemented during each cycle of seaweed availability in order to ensure sustainable process stability.

Thermodynamic efficiency of carbon capture and utilisation in anaerobic batch digestion process

Carbon capture and storage (CCS) in the oil and water industries is becoming common and a significant consumer of energy typically requiring 150–450 °C and or several hundred bar pressure [1] particularly in geological deposition. A biological carbon capture and conversion has been considered in conventional anaerobic digestion processes. The process has been utilised in biological mixed culture, where acetoclastic bacteria and hydrogenophilic methanogens play a major key role in the utilisation of carbon dioxide. However, the bio catalytic microorganisms, hydrogenophilic methanogens are reported to be unstable with acetoclastic bacteria. In this work the biochemical thermodynamic efficiency was investigated for the stabilisation of the microbial process in carbon capture and utilisation. The authors observed that a thermodynamic efficiency of biological carbon capture and utilisation (BCCU) had 32% of overall reduction in yield of carbon dioxide with complimentary increase of 30% in yield of methane, while the process was overall endothermic. Total consumption of energy (≈0.33 MJ l−1) was estimated for the carbonate solubility (0.1 mol l−1) in batched BCCU. This has a major influence on microbial composition in the bioreactor. This thermodynamic study is an essential tool to aid the understanding of the interactions between operating parameters and the mixed microbial culture.

Anaerobic digestion process for biogas and biomolecules production: microflora identification and characterization

The anaerobic process was efficient in organic matter removal. During the process, an interesting compound as quercetin was produced inside of reactor. Phylogenetic analysis showed the presence of phylotypes affiliated with gamma-Proteobacteria, Choroflexi, and Bacteroidetes. Archaea were represented by phylotypes belonging to the genus Methanosarcina and Methanosaeta.