An in vitro biofilm model system to facilitate study of microbial communities of the human oral cavity.

The human oral cavity is host to a diverse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of individual or several cultivated species, situations which do not totally reflect the function of organisms within more complex microbiota or multispecies biofilms. The number of validated models that allow examination of the role that biofilms play during oral cavity colonization is also limited. The CDC biofilm reactor is a standard method that has been deployed to study interactions between members of human microbiotas allowing studies to be completed during an extended period under conditions where nutrient availability, and washout of waste products are controlled. The objective of this work was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum to study the development, reproducibility and stability of the oral microbiota. By employing deep sequencing of the variable regions of the 16S rRNA gene, we found that the CDC biofilm reactor could be used to efficiently cultivate microbiota containing all six major phyla previously identified as the core saliva microbiota. After an acclimatisation period, communities in each reactor stabilised. Replicate reactors were predominately populated by a shared core microbiota; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units. We conclude that the CDC biofilm reactor can be used to cultivate communities that replicate key features of the human oral cavity and is a useful tool to facilitate studies of the dynamics of these communities.

Characterization and application of nonlinear plastic materials for post-CPA pulse compression.

We demonstrate the three-fold post-chirped-pulse-amplification (post-CPA) pulse compression of a high peak power laser pulse using allyl diglycol carbonate (CR39), which was selected as the optimal material for near-field self-phase modulation out of a set of various nonlinear plastic materials, each characterized with respect to its nonlinear refractive index and optical transmission. The investigated materials could be applied for further pulse compression at high peak powers, as well as for gain narrowing compensation within millijoule-class amplifiers. The post-CPA pulse compression technique was tested directly after the first CPA stage within the POLARIS laser system, with the compact setup containing a single 1 mm thick plastic sample and a chirped mirror pair, which enabled a substantial shortening of the compressed pulse duration and, hence, a significant increase in the laser peak power without any additional modifications to the existing CPA chain.

Addressing identity-related barriers to collaboration for conservation through self-affirmation theory and moral foundations theory.

The natural resource management literature documents many reasons for pursuing collaborative processes, offering useful insights on how to manage conflict and facilitate productive deliberation in complex multistakeholder collaborative efforts. Moral foundations theory and self-affirmation theory can further help collaborative efforts mitigate conflicts caused by identity threats and the identity-protective reasoning these threats provoke. Moral foundations theory suggests an approach to increase collaboration by minimizing triggering language and helping people appreciate opposing viewpoints. Self-affirmation theory suggests a practical intervention that could be used to increase collaboration by desensitizing people to identity threats and reducing defensiveness. Taken together, these theories can contribute substantially to the understanding and practice of collaboration and conflict management for conservation.

Consideración de las Barreras Relacionadas con la Identidad que Enfrenta la Colaboración para la Conservación a través de la Teoría de Autoafirmación y la Teoría de Fundamentos Morales Resumen La literatura sobre el manejo de recursos naturales documenta muchas razones por las que es necesario buscar procesos colaborativos, los cuales ofrecen conocimiento útil sobre cómo manejar el conflicto. Estos procesos también facilitan la deliberación productiva dentro de los esfuerzos colaborativos complejos en los cuales participan múltiples actores. La teoría de fundamentos morales puede ayudar a que los esfuerzos colaborativos mitiguen los conflictos causados por las amenazas a la identidad y el razonamiento de protección de identidad que estas amenazas provocan. La teoría de fundamentos morales propone una estrategia para incrementar la colaboración al minimizar el lenguaje detonante y ayudar a que las personas aprecien los puntos de vista contrarios. La teoría de autoafirmación sugiere una intervención práctica que podría usarse para incrementar la colaboración al desensibilizar a las personas a tal grado que identifiquen amenazas y reduzcan la actitud defensiva. En conjunto, estas teorías pueden contribuir sustancialmente al entendimiento y la práctica de la colaboración y el manejo de conflictos para la conservación.

A kinetic Monte Carlo approach to study fluid transport in pore networks.

The mechanism of fluid migration in porous networks continues to attract great interest. Darcy's law (phenomenological continuum theory), which is often used to describe macroscopically fluid flow through a porous material, is thought to fail in nano-channels. Transport through heterogeneous and anisotropic systems, characterized by a broad distribution of pores, occurs via a contribution of different transport mechanisms, all of which need to be accounted for. The situation is likely more complicated when immiscible fluid mixtures are present. To generalize the study of fluid transport through a porous network, we developed a stochastic kinetic Monte Carlo (KMC) model. In our lattice model, the pore network is represented as a set of connected finite volumes (voxels), and transport is simulated as a random walk of molecules, which "hop" from voxel to voxel. We simulated fluid transport along an effectively 1D pore and we compared the results to those expected by solving analytically the diffusion equation. The KMC model was then implemented to quantify the transport of methane through hydrated micropores, in which case atomistic molecular dynamic simulation results were reproduced. The model was then used to study flow through pore networks, where it was able to quantify the effect of the pore length and the effect of the network's connectivity. The results are consistent with experiments but also provide additional physical insights. Extension of the model will be useful to better understand fluid transport in shale rocks.

Trade-offs constrain the success of glyphosate-free farming.

Glyphosate, the most widely used herbicide, is linked with environmental harm and there is a drive to replace it in agricultural systems. We model the impacts of discontinuing glyphosate use and replacing it with cultural control methods. We simulate winter wheat arable systems reliant on glyphosate and typical in northwest Europe. Removing glyphosate was projected to increase weed abundance, herbicide risk to the environment, and arable plant diversity and decrease food production. Weed communities with evolved resistance to non-glyphosate herbicides were not projected to be disproportionately affected by removing glyphosate, despite the lack of alternative herbicidal control options. Crop rotations with more spring cereals or grass leys for weed control increased arable plant diversity. Stale seedbed techniques such as delayed drilling and choosing ploughing instead of minimum tillage had varying effects on weed abundance, food production, and profitability. Ploughing was the most effective alternative to glyphosate for long-term weed control while maintaining production and profit. Our findings emphasize the need for careful consideration of trade-offs arising in scenarios where glyphosate is removed. Integrated Weed Management (IWM) with more use of cultural control methods offers the potential to reduce chemical use but is sensitive to seasonal variability and can incur negative environmental and economic impacts.

Loss of coupling between calcium influx, energy consumption and insulin secretion associated with development of hyperglycaemia in the UCD-T2DM rat model of type 2 diabetes.

AIMS/HYPOTHESIS: Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes. METHODS: Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia. RESULTS: Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia. CONCLUSIONS/INTERPRETATION: These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

cJUN N-terminal kinase (JNK) activation mediates islet amyloid-induced beta cell apoptosis in cultured human islet amyloid polypeptide transgenic mouse islets.

AIMS/HYPOTHESIS: Aggregation of human islet amyloid polypeptide (hIAPP) as islet amyloid is associated with increased beta cell apoptosis and reduced beta cell mass in type 2 diabetes. Islet amyloid formation induces oxidative stress, which contributes to beta cell apoptosis. The cJUN N-terminal kinase (JNK) pathway is a critical mediator of beta cell apoptosis in response to stress stimuli including oxidative stress and exogenous application of hIAPP. We determined whether amyloid formation by endogenous hIAPP mediates beta cell apoptosis through JNK activation and downstream signalling pathways. METHODS: hIAPP transgenic and non-transgenic mouse islets were cultured for up to 144 h in 16.7 mmol/l glucose to induce islet amyloid in the presence or absence of the amyloid inhibitor Congo Red or a cell-permeable JNK inhibitor. Amyloid, beta cell apoptosis, JNK signalling and activation of downstream targets in the intrinsic and extrinsic apoptotic pathways were measured. RESULTS: JNK activation occurred with islet amyloid formation in hIAPP transgenic islets after 48 and 144 h in culture. Neither high glucose nor the hIAPP transgene alone was sufficient to activate JNK independent of islet amyloid. Inhibition of islet amyloid formation with Congo Red reduced beta cell apoptosis and partially decreased JNK activation. JNK inhibitor treatment reduced beta cell apoptosis without affecting islet amyloid. Islet amyloid increased mRNA levels of markers of the extrinsic (Fas, Fadd) and intrinsic (Bim [also known as Bcl2l11]) apoptotic pathways, caspase 3 and the anti-apoptotic molecule Bclxl (also known as Bcl2l1) in a JNK-dependent manner. CONCLUSIONS/INTERPRETATION: Islet amyloid formation induces JNK activation, which upregulates predominantly pro-apoptotic signals in both extrinsic and intrinsic pathways, resulting in beta cell apoptosis.

Exendin-4 increases islet amyloid deposition but offsets the resultant beta cell toxicity in human islet amyloid polypeptide transgenic mouse islets.

AIMS/HYPOTHESIS: In type 2 diabetes, aggregation of islet amyloid polypeptide (IAPP) into amyloid is associated with beta cell loss. As IAPP is co-secreted with insulin, we hypothesised that IAPP secretion is necessary for amyloid formation and that treatments that increase insulin (and IAPP) secretion would thereby increase amyloid formation and toxicity. We also hypothesised that the unique properties of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 to maintain or increase beta cell mass would offset the amyloid-induced toxicity. METHODS: Islets from amyloid-forming human IAPP transgenic and control non-transgenic mice were cultured for 48 h in 16.7 mmol/l glucose alone (control) or with exendin-4, potassium chloride (KCl), diazoxide or somatostatin. Human IAPP and insulin release, amyloid deposition, beta cell area/islet area, apoptosis and AKT phosphorylation levels were determined. RESULTS: In control human IAPP transgenic islets, amyloid formation was associated with increased beta cell apoptosis and beta cell loss. Increasing human IAPP release with exendin-4 or KCl increased amyloid deposition. However, while KCl further increased beta cell apoptosis and beta cell loss, exendin-4 did not. Conversely, decreasing human IAPP release with diazoxide or somatostatin limited amyloid formation and its toxic effects. Treatment with exendin-4 was associated with an increase in AKT phosphorylation compared with control and KCl-treated islets. CONCLUSIONS/INTERPRETATION: IAPP release is necessary for islet amyloid formation and its toxic effects. Thus, use of insulin secretagogues to treat type 2 diabetes may result in increased islet amyloidogenesis and beta cell death. However, the AKT-associated anti-apoptotic effects of GLP-1 receptor agonists such as exendin-4 may limit the toxic effects of increased islet amyloid.

Coupled effects of ion beam chemistry and morphology on directed self-assembly of epitaxial semiconductor nanostructures.

We study the coupled effects of ion beam chemistry and morphology on the assembly of templated epitaxial nanostructures. Using a focused ion beam (FIB) system equipped with a mass-selecting filter, we pattern Si substrates with local ion doses of Si, Ge and Ga to control subsequent Ge(x)Si(1 - x) epitaxial nanostructure assembly. This capability to employ different templating species allows us to study how different incorporated ion species in the near surface region affect the ability to localize nucleation during subsequent epitaxial growth. Our results indicate that FIB-directed self-assembly is a complex process, dependent on dose-induced morphology in addition to ion-specific chemical effects.

Making marinas bivalve friendly for enhanced biodiversity outcomes.

Natural coastlines are being replaced by artificial structures (pilings, pontoons, breakwaters), with negative environmental impacts, particularly in marinas. Ropes seeded with mussels (Mytilus galloprovincialis) were added to artificial structures in a marina, using aquaculture techniques, to reduce the colonisation of invasive taxa. After 6-months, droplines beneath pontoons had the highest seeded mussel survival and growth, richness of native and invasive taxa, and proportion of invasive to native taxa, compared with the other interventions. Mussel ropes on the intertidal structures (pilings and breakwaters) supported higher biomass of native taxa, whereas mussel ropes on subtidal structures (pontoons and breakwaters) had reduced biomass of invasive taxa, relative to the unseeded ropes. Droplines had the greater biomass of mussels, while mussel ropes placed under pontoons, and in subtidal gabion baskets limited the biomass but not the diversity of invasive species. Further study is required to determine whether these interventions can be upscaled to improve both the native biodiversity and functioning of marinas.

Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence?

Type 2 diabetes is a progressive disease characterised by islet amyloid deposits in the majority of patients. Amyloid formation is considered a significant factor in deterioration of islet function and reduction in beta cell mass, and involves aggregation of monomers of the normally soluble beta cell peptide, human islet amyloid polypeptide (hIAPP) into oligomers, fibrils and, ultimately, mature amyloid deposits. Despite extensive in vitro studies, the process of hIAPP aggregation in vivo is poorly understood, though it is widely reported to promote cytotoxicity. Recently, studies have suggested that only the early stages of fibril assembly, and in particular small hIAPP oligomers, are responsible for beta cell cytotoxicity. This challenges the prior concept that newly formed fibrils and/or mature fibrillar amyloid are cytotoxic. Herein, evidence both for and against the toxic hIAPP oligomer hypothesis is presented; from this, it is apparent that what exactly causes beta cell death when hIAPP aggregates remains debatable. Moreover, substantially more work with more specific reagents and techniques than are currently available will be required to identify conclusively the toxic species resulting from hIAPP aggregation. Keeping an open mind on the nature of the cytotoxic insult has implications for therapeutic developments and clinical care in type 2 diabetes.

The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.

The critical role of the beta cell in the pathogenesis of type 2 diabetes is now well established. When examined in patients with type 2 diabetes and individuals at increased risk, reductions in beta cell mass and abnormalities of beta cell function can both be demonstrated. The question of whether one alone is sufficient or both are necessary for the development of hyperglycaemia has been debated. Based on human and animal studies, it appears that neither alone is sufficient. Rather, for glucose to rise to the level at which diabetes would be diagnosed, defects in beta cell mass and in beta cell function are required.

Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis.

AIMS/HYPOTHESIS: Islet amyloid in type 2 diabetes contributes to loss of beta cell mass and function. Since islets are susceptible to oxidative stress-induced toxicity, we sought to determine whether islet amyloid formation is associated with induction of oxidative stress. METHODS: Human islet amyloid polypeptide transgenic and non-transgenic mouse islets were cultured for 48 or 144 h with or without the antioxidant N-acetyl-L: -cysteine (NAC) or the amyloid inhibitor Congo Red. Amyloid deposition, reactive oxygen species (ROS) production, beta cell apoptosis, and insulin secretion, content and mRNA were measured. RESULTS: After 48 h, amyloid deposition was associated with increased ROS levels and increased beta cell apoptosis, but no change in insulin secretion, content or mRNA levels. Antioxidant treatment prevented the rise in ROS, but did not prevent amyloid formation or beta cell apoptosis. In contrast, inhibition of amyloid formation prevented the induction of oxidative stress and beta cell apoptosis. After 144 h, amyloid deposition was further increased and was associated with increased ROS levels, increased beta cell apoptosis and decreased insulin content. At this time-point, antioxidant treatment and inhibition of amyloid formation were effective in reducing ROS levels, amyloid formation and beta cell apoptosis. Inhibition of amyloid formation also increased insulin content. CONCLUSIONS/INTERPRETATION: Islet amyloid formation induces oxidative stress, which in the short term does not mediate beta cell apoptosis, but in the longer term may feed back to further exacerbate amyloid formation and contribute to beta cell apoptosis.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress.

AIMS/HYPOTHESIS: Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP. METHODS: Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1). RESULTS: Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers. CONCLUSIONS/INTERPRETATION: Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

Amyloid formation results in recurrence of hyperglycaemia following transplantation of human IAPP transgenic mouse islets.

AIMS/HYPOTHESIS: Islet transplantation is a potential cure for diabetes; however, rates of graft failure remain high. The aim of the present study was to determine whether amyloid deposition is associated with reduced beta cell volume in islet grafts and the recurrence of hyperglycaemia following islet transplantation. METHODS: We transplanted a streptozotocin-induced mouse model of diabetes with 100 islets from human IAPP (which encodes islet amyloid polypeptide) transgenic mice that have the propensity to form islet amyloid (n = 8-12) or from non-transgenic mice that do not develop amyloid (n = 6-10) in sets of studies that lasted 1 or 6 weeks. RESULTS: Plasma glucose levels before and for 1 week after transplantation were similar in mice that received transgenic or non-transgenic islets, and at that time amyloid was detected in all transgenic grafts and, as expected, in none of the non-transgenic grafts. However, over the 6 weeks following transplantation, plasma glucose levels increased in transgenic but remained stable in non-transgenic islet graft recipients (p < 0.05). At 6 weeks, amyloid was present in 92% of the transgenic grafts and in none of the non-transgenic grafts. Beta cell volume was reduced by 30% (p < 0.05), beta cell apoptosis was twofold higher (p < 0.05), and beta cell replication was reduced by 50% (p < 0.001) in transgenic vs non-transgenic grafts. In summary, amyloid deposition in islet grafts occurs prior to the recurrence of hyperglycaemia and its accumulation over time is associated with beta cell loss. CONCLUSIONS/INTERPRETATION: Islet amyloid formation may explain, in part, the non-immune loss of beta cells and recurrence of hyperglycaemia following clinical islet transplantation.

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1.

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.

Control of Alopecurus myosuroides (black-grass) resistant to mesosulfuron+iodosulfuron.

Resistance to the ALS inhibitor mesosulfuron+iodosulfuron ('Atlantis') had been identified in 293 populations of A. myosuroides in the UK by 2008. Two field trials were conducted in winter wheat crops where ALS target site resistance (Pro197Thr) occurred. Mesosulfuron+iodosulfuron (12+2.4 g a.i. ha(-1)) gave 73% - 79% reductions in head numbers in Town Mead field, but only -7% - 5% reductions in Long Covert. Mixtures and sequences improved overall control. Pre-emergence flufenacet+pendimethalin (240+1200 g a.i. ha(-1)) followed by mesosulfuron+ iodosulfuron plus pendimethalin (1320 g a.i. ha(-1)) or clodinafop+trifluralin (30+960 g a.i. ha(-1)) gave 93 - 98% reductions in Town Mead but only 60 - 73% reductions in Long Covert. A non-ALS treatment of pre-emergence flufenacet+pendimethalin followed by isoproturon+pendimethalin (1500+1320 g a.i. ha(-1)) in late October and clodinafop+trifluralin in November or February achieved 97% and 93% reductions in the two trials. Seed samples collected from surviving plants were evaluated in glasshouse assays to quantify any changes in the incidence of resistance. There was an increase in the proportion of plants resistant to mesosulfuron+iodosulfuron regardless of whether it was used alone, in mixture or sequence. No such changes occured with non-ALS treatments. The trials highlight the difficulty of achieving adequate control with alternative herbicides, especially as isoproturon and trifluralin will not be available for use in the UK after 2009.

Herbicide-resistance to mesosulfuron + iodosulfuron in Alopecurus myosuroides (black-grass).

A formulated mixture of two sulfonylurea herbicides, mesosulfuron and iodosulfuron, combined with the safener mefenpyr-diethyl ('Atlantis') is being used extensively in Europe and there is concern that resistance will evolve in Alopecurus myosuroides (black-grass). Glasshouse screening bioassays showed that the best single discriminating dose for detecting resistance is the UK field rate of 12 g mesosulfuron + 2.4 g iodosulfuron ha(-1) applied at the 3 leaf stage, with herbicidal effects recorded 4 weeks later. Using this methodology with 466 UK seed samples, resistance was confirmed on a total of 24 farms in 11 counties by 2005, 81 farms in 19 counties by 2006 and 133 farms in 21 counties by 2007. Cultural histories for 10 resistant (R) and 7 susceptible (S) fields were obtained. Winter cereals were grown in 73% R/ 71% S years and a mean of 3.0 grass-weed active ingredients applied per year in both R and S fields. Four herbicide classes dominated, comprising almost 80% of all applications: ALS inhibitors 17% R/ 21% S; ACCase inhibitors 19% R/ 17% S; substituted ureas 18% R/ 10% S; dinitroanilines 25% R/ 30% S. Consequently, ALS inhibitor use was not excessive and field histories were not a reliable indicator of resistance risk. DNA sequencing of the ALS gene from resistant and susceptible individuals of nine populations was used to identify resistance mechanisms. All highly resistant individuals from seven populations showed a single nucleotide polymorphism in the first position of the Pro197 codon of an A. myosuroides ALS gene. One population showed resistant individuals with a single nucleotide polymorphism in the second position of the Trp574 codon. Consequently ALS target site resistance was confirmed in eight of the nine populations studied in detail.