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Airway smooth muscle thickening, a key characteristic of chronic asthma, is largely attributed to increased smooth muscle cell proliferation and reduced smooth muscle apoptosis. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that participates in the pathogenesis of airway smooth muscle remodeling. Although the role of Plk1 in cell proliferation and migration is recognized, its function in smooth muscle apoptosis has not been previously investigated. Caspase-9 (Casp9) is a key enzyme that participates in the execution of apoptosis. Casp9 phosphorylation at Ser-196 and Thr-125 is implicated in regulating its activity in cancer cells and epithelial cells. Here, exposure of human airway smooth muscle (HASM) cells to platelet-derived growth factorfor 24 hours enhanced the expression of Plk1 and Casp9 phosphorylation at Ser-196, but not Thr-125. Overexpression of Plk1 in HASM cells increased Casp9 phosphorylation at Ser-196. Moreover, the expression of Plk1 increased the levels of pro-Casp9 and pro-Casp3 and inhibited apoptosis, demonstrating a role of Plk1 in inhibiting apoptosis. Knockdown of Plk1 reduced Casp9 phosphorylation at Ser-196, reduced pro-Casp9/3 expression, and increased apoptosis. Furthermore, Casp9 phosphorylation at Ser-196 was upregulated in asthmatic HASM cells, which was associated with increased Plk1 expression. Knockdown of Plk1 in asthmatic HASM cells decreased Casp9 phosphorylation at Ser-196 and enhanced apoptosis. Together, these studies disclose a previously unknown mechanism that the Plk1-Casp9/3 pathway participates in the controlling of smooth muscle apoptosis.

Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott–Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin β1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin β1 and c-Abl are important for the recruitment of Abi1 to the leading edge.

There is increasing interest in the development of fortified foods enriched in menaquinone-7 as high dietary intakes may reduce the incidence of osteoporosis and cardiovascular calcification. In this work we explore the potential of using food ingredients as fermentation substrates for the development of such products. It was found that a combination of soy protein and glycerol was the most suitable for MK-7 production. The process was scaled-up; use of a dual feeding strategy was found to avoid foaming and improve MK-7 production. The MK-7 titre and productivity were 99 mg L−1 and 2.1 mg L−1 h−1 respectively, amongst the highest values reported in the open literature. Finally, the resulting product was formulated into a food (soymilk); it was found that 75% of the MK-7 remained after 24 weeks of room temperature storage. These results clearly demonstrate an approach to utilise food ingredients for the production of MK-7 enriched functional foods.

Airway smooth muscle cells require coordinated protrusion and focal adhesion dynamics to migrate properly. However, the signaling cascades that connect these two processes remain incompletely understood. Glia maturation factor (GMF)-γ has been implicated in inducing actin debranching and inhibiting nucleation. In this study, we discovered that GMFγ phosphorylation at Y104 regulates human airway smooth muscle cell migration. Using high-resolution microscopy coupled with three-dimensional object-based quantitative image analysis software, Imaris 9.2.0, phosphomimetic mutant, Y104D-GMFγ, was enriched at nascent adhesions along the leading edge where it recruited activated neural Wiskott-Aldrich syndrome protein (N-WASP; pY256) to promote actin-branch formation, which enhanced lamellipodial dynamics and limited the growth of focal adhesions. Unexpectedly, we found that nonphosphorylated mutant, Y104F-GMFγ, was enriched in growing adhesions where it promoted a linear branch organization and focal adhesion clustering, and recruited zyxin to increase maturation, thus inhibiting lamellipodial dynamics and cell migration. The localization of GMFγ between the leading edge and focal adhesions was dependent upon myosin activity. Furthermore, c-Abl tyrosine kinase regulated the GMFγ phosphorylation-dependent processes. Together, these results unveil the importance of GMFγ phosphorylation in coordinating lamellipodial and focal adhesion dynamics to regulate cell migration.

Vitamin K is essential for blood coagulation and plays important roles in bone and cardiovascular health. Menaquinone-7 (MK-7) is one form of vitamin K that is especially useful due to its long half-life in the circulation. MK-7 is difficult to make via organic synthesis, and is thus commonly produced by fermentation. This study aimed to genetically modify Bacillus subtilis cultures to increase their MK-7 yield and reduce production costs. We constructed 12 different strains of B. subtilis 168 by overexpressing different combinations of the rate-limiting enzymes Dxs, Dxr, Idi, and MenA. We observed an 11-fold enhancement of production in the best-performing strain, resulting in 50 mg/L MK-7. Metabolite analysis revealed new bottlenecks in the pathway at IspG and IspH, which suggest avenues for further optimization. This work highlights the usefulness of Bacillus subtilis for industrial production of high value compounds.

Development of a validated model of the electrodialytic treatment of bio-refinery streams would be invaluable for the techno-economic evaluation of large-scale bio-processes. Here, we outline the systematic development and validation of such a model, capable of providing accurate predictions for a range of solutions from single salts (i.e. potassium chloride at concentrations between 0.13 and 0.77 M), mixtures of simple salts (i.e. potassium and magnesium chloride) at similar concentration ranges, as well as mixtures of salts and organic compounds. In all cases, the model was capable of providing good agreement with experimentally measured concentration and voltage profiles, as well as with measured compartment volumes. Finally, we have examined the modelling of an industrial effluent (containing a complex mixture of salts and organic compounds); again the model was in good agreement with experimental data.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with N-WASP (neuronal Wiskott - Aldrich syndrome Protein), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

Vitamin K1 has recently been identified as a potentially important nutrient for prevention of chronic diseases, especially those associated with aging, like osteoporosis and cardiovascular disease. Currently the majority of Vitamin K1 is produced via chemical synthesis; the aim of this work is to examine the potential to produce Vitamin K1 using microalgae. Biological synthesis produces only the active E-isomer, whereas chemical synthesis produces 10–20% of the inactive Z-isomer. Biosynthesis also utilises mild operating conditions, sustainable production methods, and has the potential to simultaneously produce other beneficial compounds. Seven different species of microalgae were screened; the cyanobacterium Anabaena cylindrica was identified as the richest source of vitamin K1. Concentrations were of the order 200 μg g−1 on a dry-weight basis, which is around six times higher than rich dietary sources such as spinach and parsley; one gram of the algae provides approximately three times the daily adult intake for Vitamin K1. By optimizing the growth conditions (lighting and medium), the productivity was increased fourfold to 22 μg L−1 d−1. Analysis of the nutritional properties of the biomass revealed that it contained high levels of protein (69% (w/w)) and Vitamin B12 (1.5 μg g−1), in addition to the high concentration of phylloquinone. An animal study was performed to evaluate the potential toxicity of the biomass; however, no evidence of acute toxicity was observed even at relatively high addition rates (15% (w/w)). These results support the use of microalgae, specifically A. cylindrica, as a source of phylloquinone.

Proper arteriovenous morphogenesis is crucial for maintaining normal tissue perfusion. However, our understanding of how arterial morphogenesis is regulated in the CNS is incomplete. In this study, we asked whether vascular basement membrane (BM) laminins, specifically the γ3-containing isoforms, regulate retinal arterial morphogenesis. We provide evidence that Laminin-γ3 is deposited at both arterial and venous BMs during arteriogenesis. Vascular BM Laminin-γ3 bound dystroglycan (DG), a laminin receptor preferentially expressed by arterial endothelial cells (ECs) during arteriogenesis. Blockade of laminin-DG binding in vitro led to decreased Delta-like ligand (DLL)-4 expression in ECs. Moreover, genetic deletion of the Laminin-γ3- and EC-specific deletion of DG led to similar defects in retinal arteriogenesis, including reduced Dll4 expression, hyperbranching and reduced smooth muscle coverage. These results implicate a newly identified Laminin-γ3-DG signaling cascade that regulates arterial Dll4/Notch signaling to specify and stabilize retinal arteries.-Biswas, S., Watters, J., Bachay, G., Varshney, S., Hunter, D. D., Hu, H., Brunken, W. J. Laminin-dystroglycan signaling regulates retinal arteriogenesis.

The primary objective of this review is to propose an approach for the biosynthesis of phylloquinone (vitamin K1) based upon its known sources, its role in photosynthesis and its biosynthetic pathway. The chemistry, health benefits, market, and industrial production of vitamin K are also summarized. Vitamin K compounds (K vitamers) are required for the normal function of at least 15 proteins involved in diverse physiological processes such as coagulation, tissue mineralization, inflammation, and neuroprotection. Vitamin K is essential for the prevention of Vitamin K Deficiency Bleeding (VKDB), especially in neonates. Increased vitamin K intake may also reduce the severity and/or risk of bone fracture, arterial calcification, inflammatory diseases, and cognitive decline. Consumers are increasingly favoring natural food and therapeutic products. However, the bulk of vitamin K products employed for both human and animal use are chemically synthesized. Biosynthesis of the menaquinones (vitamin K2) has been extensively researched. However, published research on the biotechnological production of phylloquinone is restricted to a handful of available articles and patents. We have found that microalgae are more suitable than plant cell cultures for the biosynthesis of phylloquinone. Many algae are richer in vitamin K1 than terrestrial plants, and algal cells are easier to manipulate. Vitamin K1 can be efficiently recovered from the biomass using supercritical carbon dioxide extraction.

Poly(β-hydroxybutyrate) (PHB) thermoplastics hold appeal for many applications due to their high biodegradability compared to petrochemical-based plastics. PHB is currently produced using fermentation technologies and purification techniques that have considerable potential for optimization. Current PHB production methods require impractical amounts of organic solvents for the removal of residual fermentation media necessary for medical utility. We conceptualized an alternative process using high-pressure CO2 as an alternative solvent for PHB purification. The extraction of plant oil residues that are the main contaminants in PHB produced by fermentation were compared under conditions of varying pressure, temperature, and co-solvents. Using pure CO2 at 150 bar and 50 °C achieved the removal of 73 ± 5 wt% of oil residue, and this was increased to 93 ± 3 wt% using an ethanol–CO2 expanded solution. This novel approach to the purification of PHB will enable further developments of PHB thermoplastics for biomedical applications.

Electrodialysis (ED) has the potential to cost-effectively separate ions from organics in bio-refinery effluents, thereby simplifying downstream treatment and producing streams for possible valorisation. However, there is little information in the open literature regarding the impact of membrane selection for such systems. In this study, we have examined the effect of ion exchange membrane selection on key performance parameters (i.e. desalination rate, specific electrical energy consumption and the transfer of organics) using three commercially available anion and cation exchange membranes with bio-refinery effluents. It was found that the choice of anion exchange membrane had a significant impact, particularly after depletion of those anions having a high membrane permeability (e.g. chloride). The apparent stack resistance (and therefore the specific electrical energy consumption) was found to depend on the choice of anion exchange membrane (from 68 Ω for a PCSA/CMX membrane pair to 188 Ω for an AHA/CMX membrane pair with a cane molasses effluent); additionally the apparent stack resistance was also closely correlated with the amount of organics crossing the membrane. Collectively, these results show that careful membrane selection is critical, particularly when a high desalination rate is desired.

Fucoxanthin is a carotenoid pigment produced by algae that has a range of potential health benefits. Despite the obvious interest in developing a process for the production of fucoxanthin, relatively few authors have systematically examined the impact of culture conditions (i.e. the light intensity, medium composition and CO2 addition) on fucoxanthin production. In this work, we have addressed this issue using the marine microalgae Phaeodactylum tricornutum. It was found that at low light intensities (100 μmol photons m− 2 s− 1) the specific fucoxanthin concentration was greater (42.8 ± 19.5 mg g− 1) than at a higher intensity of 210 μmol photons m− 2 s− 1 (9.9 ± 4.2 mg g− 1). Addition of nitrate to the medium led to a significant increase in the specific fucoxanthin concentration with the maximum specific concentration (59.2 ± 22.8 mg g− 1), volumetric concentration (20.5 mg L− 1) and bioreactor productivity (2.3 mg L− 1 day− 1) being observed with the nitrate enriched medium. These reproducible results from our systematic investigation into the effect of culture conditions on fucoxanthin production are highly encouraging and clearly demonstrate the potential for P.tricornutum to be employed as a natural source of fucoxanthin in nutraceutical applications.

Retinal vascular diseases are among the leading causes of acquired blindness. In recent years, retinal microglia have been shown to influence vascular branching density and endothelial cell proliferation. However, how microglial recruitment and activation are regulated during development remains unclear. We hypothesized that microglial recruitment, activation, and down-stream signaling are modulated by components of the mural basement membrane. We used a reverse genetic approach to disrupt laminin expression in the vascular basement membrane and demonstrate that microglia respond to the mural basement membrane in an isoform-specific manner. Microglial density is significantly increased in the laminin γ3-null (Lamc3−/−) retinal superficial vascular plexus and consequently the vascular branching density is increased. Microglia also respond to astrocyte-derived matrices and become hyperactivated in the Lamc3−/− retina or when tested in vitro with cell-derived matrix. Pharmacological activation of microglia in the wild-type retina produced an Lamc3−/−-like vascular phenotype, whereas pharmacological blocking of microglial activation in the Lamc3−/− retina rescued the wild-type vascular phenotype. On the molecular level, microglial transforming growth factor-β1 expression is down-regulated in the Lamc3−/− retina, and SMAD signaling decreased in endothelial cells with a consequent increase in endothelial proliferation. The reverse effects were seen in the Lamb2−/− retina. Together, our results demonstrate a novel mechanism by which laminins modulate vascular branching and endothelial cell proliferation during retinal angiogenesis.

Modern bio-refineries generate considerable volumes of wastewater that are highly coloured (5600–572,000 PtCo) as well as being concentrated in both salts (conductivities up 72.4 mS cm− 1) and organics (up to 380 g COD L− 1). In this study, bench-scale batch electrodialysis was performed to examine the feasibility of separating salts from organics for a range of industrial biorefinery streams. High levels of salt removal (up to 96% for a lignocellulosic effluent) were possible with minimal organic losses (0.3–6.3%), while key performance parameters were highly encouraging (current efficiencies = 69–104%; specific power consumption = 0.44–1.59 kWh kg− 1 of salt recovered). Collectively, the experimental results obtained here showed the cost-effective potential of electrodialysis to separate salts from organics in complex bio-refinery streams at the industrial scale.

The separation of americium(III) from europium(III) was achieved utilizing a bis-2,6-(5,6,7,8-tetrahydro-5,9,9-trimethyl-5,8-methano-1,2,4-benzotriazin-3-yl) pyridine (CA-BTP) chromatographic resin. The extraction chromatographic materials were prepared using various concentrations of CA-BTP. This new, hydrolytically stable extractant was impregnated on an inert polymeric support at 40% loading. The uptake of Am(III) and Eu(III) by this material from 0.1 to 4.0 M aqueous HNO3 solutions was measured. The resulting dry weight distribution ratios, D w , indicated a strong preference for Am(III) with little affinity for Eu(III). These results are similar to recently reported solvent extraction studies indicating a maximum uptake of Am(III) in the 0.5–1.0 M HNO3 range. The resin preparation, performance, and characterization of the Am/Eu separation are reported herein.

The development of liquid–liquid separation processes for the effective removal of minor actinide Am(III) from used nuclear fuel (UNF) using ligand-based strategies continues to be an area of signi...

As part of an on-going research program into the development of accurate computational fluid dynamics models of industrial bubble column bio-reactors, the behaviour of a range of commercially available antifoaming compounds was investigated. Experimental data from a laboratory scale system showed that increasing the antifoam concentration led to a decrease in the Oxygen Transfer Rate (OTR) up to a critical value, hypothesised to be related to the monolayer coverage of the gas-liquid interface; further increases in antifoam concentration had no additional impact. Beyond this critical antifoam concentration, a 3–5 fold reduction in the OTR was found, this reduction being independent of the type of antifoam used or the superficial air velocity. The issue of antifoam ‘deactivation’ was also examined with the results obtained being consistent with the deactivation of polyethylene oxide type antifoams being caused by their displacement from the gas-liquid interface by more hydrophobic material.

Inflammation-resolution is a protective response that is mediated by specialized pro-resolving mediators (SPMs). The clearance of dead cells or efferocytosis is a critical cellular program of inflammation-resolution. Impaired efferocytosis can lead to tissue damage in prevalent human diseases, like atherosclerosis. Therefore understanding mechanisms associated with swift clearance of dead cells is of utmost clinical importance. Recently, the accumulation of necroptotic cells (NCs) was observed in human plaques and we postulated that this is due to defective clearance programs. Here we present evidence that NCs are inefficiently taken up by macrophages because they have increased surface expression of a well-known “don’t eat me” signal called CD47. High levels of CD47 on NCs stimulated RhoA-pMLC signaling in macrophages that promoted “nibbling”, rather than whole-cell engulfment of NCs. Anti-CD47 blocking antibodies limited RhoA-p-MLC signaling and promoted whole-cell NC engulfment. Treatment with anti-CD47 blocking antibodies to Ldlr(−/−) mice with established atherosclerosis decreased necrotic cores, limited the accumulation of plaque NCs and increased lesional SPMs, including Resolvin D1 (RvD1) compared with IgG controls. Mechanistically, RvD1 promoted whole-cell engulfment of NCs by decreasing RhoA signaling and activating CDC42. RvD1 specifically targeted NCs for engulfment by facilitating the release of the well-known “eat me signal” called calreticulin from macrophages in a CDC42 dependent manner. Lastly, RvD1 enhanced the clearance of NCs in advanced murine plaques. Together, these results suggest new molecules and signaling associated with the clearance of NCs, provide a new paradigm for the regulation of inflammation-resolution, and offer a potential treatment strategy for diseases where NCs underpin the pathology.

Escherichia coli O157:H7 remains a public health problem in the United States despite a dramatic increase in the awareness of, and concern about, foodborne infections since the 1993 multistate E. coli O157:H7 epidemic. Although surveillance data can be difficult to interpret, the incidence of endemic disease caused by this organism is probably not increasing, and might be decreasing, at least in selected populations. With increased recognition of E. coli O157:H7 infection has come the investigation of increasing number of outbreaks, leading to the recognition of many "new" vehicles, including some foods not traditionally associated with enteric infections, such as dry-cured salami and lettuce. Molecular fingerprinting techniques are being used to track the transmission of E. coli O157:H7 through human populations. Analysis of DNA encoding virulence factors and surface antigens suggests that diarrheagenic E. coli have evolved by acquiring large DNA fragments, with subsequent chromosomal recombination. Some Shiga toxin-producing E. coli other than E. coli O157:H7 are no doubt pathogens, but the majority of these toxigenic strains found in food are probably not virulent. More research is needed to define the characteristics that render selected Shiga toxin-producing organisms harmful to humans.

Allergic asthma is characterized by airway smooth muscle layer thickening, which is largely attributed to cell division that requires the formation of centrosomes. Centrosomes play a pivotal role in regulating bipolar spindle formation and cell division. Before mitosis, centrosomes undergo maturation characterized by expansion of pericentriolar material proteins, which facilitates spindle formation and mitotic efficiency of many cell types. Although polo-like kinase 1 (Plk1) has been implicated in centrosome maturation, the mechanisms by which Plk1 regulates the cellular process are incompletely elucidated. Here, we identified paxillin as a new Plk1-interacting protein in human airway smooth muscle cells. We unexpectedly found that phosphorylated paxillin (Ser-272) was localized in centrosomes of human smooth muscle cells, which regulated centrosome maturation and spindle assembly. Plk1 knockdown inhibited paxillin Ser-272 phosphorylation, centrosome maturation, and cell division. Furthermore, exposure to allergens enhanced airway smooth muscle layer and paxillin phosphorylation at this residue in mice, which was reduced by smooth muscle conditional knockout of Plk1. These findings suggest that Plk1 regulates centrosome maturation and cell division in part by modulating paxillin phosphorylation on Ser-272. Furthermore, Plk1 contributes to the pathogenesis of allergen-induced thickening of the airway smooth muscle layer by affecting paxillin phosphorylation at this position.

Zeaxanthin is a carotenoid pigment used in the food industry as well as in supplements for age-related macular degeneration. There is potential for microalgae to be used for zeaxanthin production, however there is relatively little work examining this issue, particularly with respect to scale-up. Here two species of cyanobacteria (Synechococcus sp. PCC7002 and Synechocystis sp. PCC6803) were examined, along with a Rhodophyte (Rhodosorus sp.). At a light intensity of 80 μmol photons m−2 s−1 specific zeaxanthin contents were 2.30 ± 0.84, 1.61 ± 0.68 and 2.16 ± 0.63 mg g−1 for Synechococcus sp. PCC7002, Synechocystis sp. PCC6803 and Rhodosorus sp., respectively. Of the species examined the Synechococcus PCC7002 had the highest specific growth rate (0.74 ± 0.09 day−1). This species was used to further optimize the process, increasing the nutrient concentration in the medium and using an incremental light addition strategy led to an approximately 13-fold increase in the cell density, giving a final dry cell weight of 4.25 ± 1.42 g L−1. Results from this work are among the highest in the literature for zeaxanthin productivity (0.7 ± 0.5 mg L−1 day−1) and the approach used can be readily scaled-up. Additionally, the approach used in this work to achieve high cell densities of PCC7002 could be potentially applied to the production of other compounds (e.g. phycobilins or compounds produced using metabolic engineering).

Complete degradation of mixtures of organic pollutants is a major challenge due to their diverse degradation pathways. In this work, a novel microplasma bubble (MPB) reactor was developed to generate plasma discharges inside small forming bubbles as an effective mean of delivering reactive species for the degradation of the target organic contaminants. The results show that the integration of plasma and bubbles resulted in efficient degradation for all azo, heterocyclic, and cationic dyes, evidenced by the outstanding energy efficiency of 13.0, 18.1 and 22.1 g/kWh with 3 min of processing, in degrading alizarin yellow (AY), orange II (Orng-II) and methylene blue (MB), individually. The MPB treatment also effectively and simultaneously degraded the dyes in their mixtures such as AY + Orng-II, AY + MB and AY + Orng-II + MB. Scavenger assays revealed that the short-lived reactive species, including the hydroxyl ( OH) and superoxide anion ( O2−) radicals, played the dominant role in the degradation of the pollutants. Possible degradation pathways were proposed based on the intermediate products detected during the degradation process. The feasibility of this proposed strategy was further evaluated using other common water pollutants. Reduced toxicity was confirmed by the observed increases in human cell viability for the treated water. This work could support the future development of high performance- and energy-efficient wastewater abatement technologies.

There is increasing interest in the development of in situ product recovery technologies for bioprocesses as they have the potential to improve both process performance and economics. The most widely used technique is liquid-liquid extraction. However, relatively little hydrodynamic data about three-phase (gas-organic-aqueous) bubble columns are available. In this work we have examined the effect of three oil phases on the hold-up, bubble size distribution and droplet size distribution at oil loadings between 0.01 and 5% (v/v) and superficial velocities between 0.01 and 0.10 m/s. It was found that the oil phase was evenly dispersed throughout the column, addition of the oil led to a reduction in the overall hold-up by up to 34%. The recovery of three terpenoid compounds (limonene, nerolidol and β-carotene) was examined using 1% (v/v) hexadecane as the extraction phase. It was found that the organic phase could be separated using a simple gravity separator; product recoveries ranged from 66 to 95% for an air/water system. 5 g L−1 molasses was added to mimic fermentation medium, this led to an approximately 10% reduction in the observed recovery. The approach used concentrates the product by a factor of approximately 100, is mechanically simple and can be readily adapted to continuous operation, which are advantages from a scale-up perspective. Results from this work can be used in the design and scale-up of in situ product recovery technologies for bubble column bioreactors.

Vimentin intermediate filaments (IFs) link to desmosomes (intercellular junctions) on the membrane and dense bodies in the cytoplasm, which provides a structural base for intercellular and intracellular force transmission in smooth muscle. There is evidence to suggest that the vimentin framework plays an important role in mediating smooth muscle mechanical properties such as tension and contractile responses. Contractile activation induces vimentin phosphorylation at Ser-56 and vimentin network reorientation, facilitating contractile force transmission among and within smooth muscle cells. p21-activated kinase 1 and polo-like kinase 1 catalyze vimentin phosphorylation at Ser-56, whereas type 1 protein phosphatase dephosphorylates vimentin at this residue. Vimentin filaments are also involved in other cell functions including migration and nuclear positioning. This review recapitulates our current knowledge how the vimentin network modulates mechanical and biological properties of smooth muscle.

Polo-like kinase 1 (Plk1) has been implicated in mitosis, cytokinesis, and proliferation. The mechanisms that regulate Plk1 expression remain to be elucidated. It is reported that miR-100 targets Plk1 in certain cancer cells. Here, treatment with miR-100 did not affect Plk1 protein expression in human airway smooth muscle cells. In contrast, treatment with miR-509 inhibited the expression of Plk1 in airway smooth muscle cells. Exposure to miR-509 inhibitor enhanced Plk1 expression in cells. Introduction of miR-509 reduced luciferase activity of a Plk1 3′UTR reporter. Mutation of miR-509 targeting sequence in Plk1 3′UTR resisted the reduction of the luciferase activity. Furthermore, miR-509 inhibited the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation without affecting the expression of c-Abl, a tyrosine kinase implicated in cell proliferation. Moreover, we unexpectedly found that vimentin filaments contacted paxillin-positive focal adhesions. miR-509 exposure inhibited vimentin phosphorylation at Ser-56, vimentin network reorganization, focal adhesion formation, and cell migration. The effects of miR-509 on ERK1/2 and vimentin were diminished in RNAi-resistant Plk1 expressing cells treated with miR-509. Taken together, these findings unveil previously unknown mechanisms that miR-509 regulates ERK1/2 and proliferation by targeting Plk1. miR-509 controls vimentin cytoskeleton reorganization, focal adhesion assembly, and cell migration through Plk1.

Vertebrate retinal development follows a pattern during which retinal progenitor cells (RPCs) give rise to all retinal cell types in a highly conserved temporal sequence. RPC proliferation and cell cycle exit are tightly coordinated to ensure proper and timely production of each of the retinal cell types. Extracellular matrix (ECM) plays an important role in eye development, influencing RPC proliferation and differentiation. In this study, we demonstrate that laminins, key ECM components, in the inner limiting membrane, control mitotic spindle orientation by providing environmental cues to the RPCs. In vivo deletion of laminin β2 in mice of both sexes results in a loss RPC basal processes and contact with the ECM, leading to a shift of the mitotic spindle pole orientation toward asymmetric cell divisions. This leads to decreased proliferation and premature RPC pool depletion, resulting in overproduction of rod photoreceptors at the expense of bipolar cells and Muller glia. Moreover, we show that deletion of laminin β2 leads to disruption and mislocalization of its receptors: dystroglycan and β1-integrin. Addition of exogenous β2-containing laminins to laminin β2-deficient retinal explants stabilizes the RPC basal processes and directs their mitotic spindle orientation toward symmetric divisions, leading to increased RPC proliferation, as well as restores proper receptor localization at the retinal surface. Finally, functional blocking of dystroglycan in wild-type retinal explants phenocopies laminin β2 ablation. Our data suggest that dystroglycan-mediated signaling between RPCs and the ECM is of key importance in controlling critical developmental events during retinogenesis.SIGNIFICANCE STATEMENT The mechanisms governing retinogenesis are subject to both intrinsic and extrinsic signaling cues. Although the role of intrinsic signaling has been the subject of many studies, our understanding of the role of the microenvironment in retinal development remains unclear. Using a combination of in vivo and ex vivo approaches, we demonstrate that laminins, key extracellular matrix components, provide signaling cues that control retinal progenitor cell attachment to the basement membrane, mitotic axis, proliferation, and fate adoption. Moreover, we identify, for the first time, dystroglycan as the receptor responsible for directing retinal progenitor cell mitotic spindle orientation. Our data suggest a mechanism where dystroglycan-mediated signaling between the cell and the extracellular matrix controls the proliferative potential of progenitors in the developing CNS.

In this study, the oxygen transfer rate (OTR), bubble size distribution, interfacial area per unit volume and liquid film mass transfer coefficient have all been measured in a 0.39 m diameter pilot-scale bubble column operated at industrially relevant superficial velocities between 0.14 and 0.28 m s−1 for which few data exist in the literature. The measured OTR ranged from 6.5 to 8.8 kg m−3 h−1, with values generally increasing with superficial velocity. The mean bubble size in the column was 3–5 mm, giving an interfacial area per unit volume for mass transfer of some 380–570 m2 m−3. Experimental data were used to validate a computational fluid dynamics (CFD) based model of the bubble column; it was found that this model could predict the experimentally measured OTR within 20% across the range of superficial velocities examined.

In this work we have quantified the effect of surfactant addition on the overall hold-up, local hold-up, liquid velocity, gas velocity, bubble size distribution (BSD) and mixing time using a bubble column 0.39 m in diameter and 2 m in height operated at superficial velocities between 0.01 and 0.28 m/s. The surfactant used was 2-propanol, which was chosen because its addition causes an air/water system to behave similarly to the complex and poorly defined fermentation media used in industrial bioprocesses. The second portion of the study examines the extension of an existing computational fluid dynamics (CFD) model to account for the impact of surfactant addition; it was found that the inclusion of a correction term into the drag coefficient calculation led to predictions that were in good agreement with experimental results.

We have examined the effect of superficial velocity, tracer injection location and measurement location on the mixing time in a bubble column. It was found that for the range of superficial velocities examined (0.07–0.29 m/s) there was little change in mixing time with superficial velocity. In contrast, it was found that the tracer injection point and measurement location had a large impact on the measured mixing time, with measured values ranging between 3 s and 25 s. Additionally, it was found that it was possible to introduce the tracer in such a way as to produce zones of poor mixing, leading to a high local tracer concentration. Such a finding is important in the design of bubble columns used in the bio-processing industry as these conditions can lead to a reduction in the yield of industrial bioprocesses. The second half of this work examines the use of Computational Fluid Dynamics (CFD) as a tool to model mixing in bubble columns. It was found that a computationally efficient CFD model correctly predicted the trends in mixing time for all superficial velocities and tracer addition locations examined, with the model being able to correctly predict the mixing time within the margin of variation.

Century old mine tailings in the Cobalt and Silver Center areas are widely dispersed throughout the terrestrial and aquatic environments and contain high concentrations of arsenic. Arsenic concentrations were found to be as high as 972 μg/L in surface waters and 10,800 mg/kg in lake sediment. The mean values for arsenic in surface waters and sediment from 9 lakes directly influenced by mining activity were 431 μg/L and 1704 mg/kg, respectively, whereas in the 12 control lakes with no mining activity in their catchment had mean values of 2.2 μg/L and 11 mg/kg in their water and sediment, respectively. Lakes impacted by downstream tailing migration (n = 4) were also assessed and had intermediate concentrations of arsenic. Principal component analysis identified contaminated lakes as having different geochemical signatures than control lakes but lake sediment that was sampled below tailings in contaminated lakes, deposited pre-mining, can resemble the geochemistry of those found in control lakes. Arsenic concentrations in these samples ranged from 4.4 to 185 mg/kg, which can be considered reasonable background as these areas contained abundant mineral deposits that could naturally elevate background concentrations. Even though background concentrations are naturally elevated, the presence of arsenic-rich tailings in these lakes has prevented any natural recovery from occurring. Fe-Mn oxides at the water-sediment interface perpetually scavenge arsenic from buried tailings below and from contaminated surface waters that cause arsenic concentrations to remain enriched in the upper sediments even after tailings have been buried by lake sediment. This process has prevented recovery of the lake ecosystems even after nearly a century without mining.

During cell migration a critical interdependence between protrusion and focal adhesion dynamics is established and tightly regulated through signaling cascades. Here we demonstrate that c-Abl, a non-receptor tyrosine kinase, can control these migratory structures through the regulation of two actin-associated proteins, glia maturation factor-γ (GMFγ) and Neural Wiskott-Aldrich syndrome protein (N-WASP). Phosphorylation of GMFγ at tyrosine-104 by c-Abl directs activated N-WASP (pY256) to the leading edge, where it can promote protrusion extension. Non-phosphorylated GMFγ guides N-WASP (pY256) to maturing focal adhesions to enhance further growth. Antagonizing this signaling pathway through knockdown or mutation of tyrosine-104 to its non-phosphorylated form attenuates migration, whereas the phospho-mimic mutant GMFγ enhances migration, thus demonstrating c-Abl, GMFγ, and activated N-WASP (pY256) as a critical signaling cascade for regulating migration in a primary human cell line.

Background Airway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an “engine” (myosin activation) and a “transmission system” (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated. Methods The association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction. Results Stimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation). Conclusions Our results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.

The following text is an additional technical note for: Impact of Surfactant Addition on Oxygen Mass Transfer in a Bubble Column by Dale D. McClure, Ai Chia Lee, John M. Kavanagh, David F. Fletcher, and Geoffrey W. Barton Chem. Eng. Technol. 2015, 38 (1), 44–52. DOI: 10.1002/ceat.201400403.

Vimentin is a major intermediate filament protein in vascular endothelial cells which might be involved in their function as a barrier tissue. It is proposed to dynamically maintain integrity of the endothelium as a tightly regulated permeability barrier that is subjected to a variety of shear and contractile forces. The results described in this report demonstrate that vimentin plays that role through mechanisms that are dependent on its phosphorylation state. Withaferin A (WFA), a vimentin targeting drug is shown to disrupt endothelial barrier function through its effects on vimentin filament distribution and physical properties. These effects are related to WFA's ability to increase vimentin phosphorylation. Through overexpressing a non-phosphorylatable vimentin mutant we can block the effects of WFA on vimentin distribution and barrier permeability. The barrier augmentation effect appears to extend to endothelial cells that do not express detectable mutant vimentin which might suggest transmissible effects across cells. Blocking vimentin phosphorylation also protects the endothelial barrier against LPS endotoxin, implicating it as a target for drug development against pulmonary edema and acute respiratory distress syndrome (ARDS).

Lutein is a carotenoid pigment which has applications in the food and nutraceutical sectors, both for its use as a colourant and for its applications as a nutraceutical. Microalgae are a promising source of lutein, however to meet market demand there is a need to develop technologies which can used for large-scale production. The effect of process conditions at a 5 L scale were quantified, it was found that at low light intensities (160 μmol photons m−2 s−1) the measured specific lutein concentration was 7.4 ± 2.1 mg g−1 which was greater than the concentration of 3.1 ± 1.1 mg g−1 obtained at higher (440 μmol photons m−2 s−1) light intensities. Supplementation of the medium with nitrate led to significant increases in the maximum specific lutein concentration (10.4 ± 5.5 mg g−1) compared to medium without nitrate addition (4.3 ± 2.9 mg g−1). These conclusions were used to scale-up the process using 50 L bubble column photo-bioreactors, it was found that the productivity was maximised (1.6 mg L−1 day−1) using an air flow-rate of 1 vvm and 24 h lighting. Finally, it was found that the process could be operated on a semi-continuous basis for 32 days, achieving high lutein concentrations (15–20 mg L−1) with the performance of the system being consistent for this duration. Results from this work have clear application in the development of large-scale systems for the microalgal production of lutein.

Phylloquinone (vitamin K1) is an essential nutrient for both humans and animals; it plays a key role in blood coagulation amongst other processes. In this work we describe the scale-up of a biosynthetic process for phylloquinone production using the cyanobacterium Anabaena cylindrica. A 50 L bubble column photo-bioreactor was used to cultivate A. cylindrica and the effects of a wide range of operating conditions including superficial velocities (0.67–6.7 cm s−1), day lengths (12–24 h), sparger designs and medium compositions were examined. The column design and superficial velocity had minimal impact on phylloquinone production, while changes to the medium composition and day length had large impacts. By varying these factors we were able to achieve final phylloquinone titres of the order 280 μg L−1 and productivities of 40 μg L−1 day−1, which are approximately double the values previously obtained. The biomass produced has clear applications in human and animal nutrition as its phylloquinone concentration was approximately 330 μg g−1 (dry basis); this being ten times higher than rich dietary sources. Conclusions regarding the effect of photo-bioreactor design and operating conditions can be applied to the production of phylloquinone, as well as the scale-up of cyanobacterial cultures more broadly.

The impact of surfactant addition on the overall holdup, drag coefficient, and bubble size distribution (BSD) in a bubble column operating at industrially relevant superficial velocities was analyzed. A range of surfactants was examined, including alcohols, sugars, and antifoaming agents. It was ascertained that the effect of a surfactant on the holdup and BSD could be related to the chemical structure of the compound, specifically its hydrophilic/hydrophobic nature as characterized by the octanol/water partition coefficient. Addition of 2-propanol to an air/water system induces a behavior similar to the fermentation media used in aerobic bioprocesses, meaning that such a system can be applied as a meaningful physical analogue in pilot-scale experimentation.

Studies have shown a much enhanced differentiation between trivalent actinides and trivalent lanthanides when substituted alkyl dithiophosphinic acids are utilized as the active phase transfer reagents. We present a study evaluating the use of a synergistic combination of bis(o-trifluoromethylphenyl)dithiophosphinic acid and trioctylphosphine oxide for the complete actinide recovery from UREX-like raffinates. A complete single-step separation of uranium, neptunium, plutonium, and americium from aqueous mixtures containing 0.5 M nitric acid and a total fission product content of 5.5 g/L (45 mM) has been demonstrated.

Pathologies of retinal blood vessels are among the major causes of blindness worldwide. A key cell type that regulates retinal vascular development is the astrocyte. Generated extrinsically to the retina, astrocytes migrate into the retina through the optic nerve head. Even though there is a strong correlation between astrocyte distribution and retinal vascular development, the factors that guide astrocytes into the retina remain unclear. In this study, we show that astrocytes migrate within a laminin-containing basement membrane - the inner limiting membrane. Genetic deletion of the laminin β2 and γ3 chains affects astrocyte migration and spatial distribution. We show that laminins act as haptotactic factors in vitro in an isoform-specific manner, inducing astrocyte migration and promoting astrocyte differentiation. The addition of exogenous laminins to laminin-null retinal explants rescues astrocyte migration and spatial patterning. Furthermore, we show that the loss of laminins reduces β1 integrin expression in astrocytes. Culturing laminin-null retinal astrocytes on laminin substrates restores focal localization of β1 integrin. Finally, we show that laminins containing β2 and γ3 chains regulate subsequent retinal blood vessel growth and maintain vascular integrity. These in vivo and in vitro studies demonstrate clearly that laminins containing β2 and γ3 chains are indispensable for migration and spatial organization of astrocytes and that they play a crucial role during retinal angiogenesis in vivo.

Background The intermediate filament protein vimentin undergoes reversible phosphorylation and dephosphorylation at Ser-56, which plays an important role in regulating the contraction-relaxation cycles of smooth muscle. The protein phosphatases that mediate vimentin dephosphorylation in smooth muscle have not been previously investigated. Methods The associations of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with vimentin in mouse tracheal rings was evaluated by co-immunoprecipitation. Lentivirus-mediated shRNA against PP1 was used to assess the role of PP1 in vimentin dephosphorylation and the vimentin-associated process in smooth muscle. Results Co-immunoprecipitation analysis showed that vimentin interacted with PP1, but barely with PP2A, in airway smooth muscle. Knockdown of PP1 by lentivirus-mediated shRNA increased the acetylcholine-induced vimentin phosphorylation and smooth muscle contraction. Because vimentin phosphorylation is able to modulate p130 Crk-associated substrate (p130CAS) and actin polymerization, we also evaluated the role of PP1 in the biological processes. Silencing of PP1 also enhanced the agonist-induced the dissociation of p130CAS from vimentin and F/G-actin ratios (an index of actin polymerization). However, PP1 knockdown did not affect c-Abl tyrosine phosphorylation, an important molecule that controls actin dynamics. Conclusions Taken together, these findings suggest that PP1 is a key protein serine/threonine phosphatase that controls vimentin Ser-56 dephosphorylation in smooth muscle. PP1 regulates actin polymerization by modulating the dissociation of p130CAS from vimentin, but not by affecting c-Abl tyrosine kinase.

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr412 (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G0/G1 to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

Most plant production by emergent coastal marshes occurs belowground. This belowground production adds to the accumulation of organic matter sustaining salt marshes as sea level rises, thus preventing excessive flooding, eventual plant death, and habitat loss. The ubiquitous nutrient enrichment of coastal salt marshes stimulating aboveground plant growth may result in higher rates of inorganic matter accumulation that compensates for marsh flooding caused by sea level rise. Results from several short-term experiments, however, demonstrate that root and rhizome biomass and carbon accumulation is reduced with nutrient enrichment, suggesting that eutrophication of coastal waters may not be a compensatory counterbalance to the effects of global sea level rise on salt marshes. We show that the net effects of 36 yr of nutrient enrichment in replicated field experiments do not lead to higher organic or inorganic accumulation. Enrichment reduces organic matter belowground and may result in a significant loss in marsh elevation equivalent to about half the average global sea level rise rates. Sustaining and restoring coastal emergent marshes is more likely if they receive less, not more, nutrient loading.

The purpose of this study was to evaluate the physiologic and perceptual responses to a continuous functional exercise workout. Ten men and 10 women (21.2 +/- 2.4 and 21.0 +/- 1.5 years) completed a maximal oxygen uptake test, strength test, and body composition analysis. Subjects then participated in 3 familiarization sessions, during which they followed a videotaped routine that consisted of a series of functional resistance exercises performed in a continuous manner. Subjects performed the same routine in a final session, during which VO2, VCO2, respiratory exchange ratio (RER), RPE, and heart rate were measured and blood samples were taken and analyzed for blood lactic acid concentration. Descriptive statistics were calculated for RPE, RER, blood lactic acid concentration, energy expenditure, and absolute and relative oxygen uptake and heart rate. Energy expenditure was calculated using VO2 and RER. Independent t-tests were used to examine differences between men and women for oxygen consumption, weight lifted, and energy expenditure during the workout. Subjects had a mean VO2 of 27.8 ml.kg.min (51% of VO2 peak and 47.8% of VO2 reserve), a mean heart rate of 156 bpm (83% of maximum heart rate), and a mean RER of 0.91. The mean RPE was 5.9, and the mean difference between pre and post lactic acid concentration was 2.5 mmol.L. The mean total caloric expenditure was 289 kcal. Men lifted significantly heavier weights and expended more total calories than women. Caloric expenditure (kcal x kg x min), VO2, and weight lifted were similar between men and women when expressed relatively. Performing dynamic functional exercises in a continuous manner resulted in energy expenditure values, but not relative VO2 values, that meet the American College of Sports Medicine recommendations.