Search

Tephritid fruit flies are susceptible to insecticide treatments when leaving infested fruit to pupate in the soil and when emerging as adults. Laboratory experiments involved placing third instar larvae of the Mexican fruit fly, Anastrepha ludens on sand treated with the naturally-derived insecticide spinosad (SpinTor 12SC). Negative correlations were detected between the concentration of spinosad and pupation and adult emergence. Treatment of pupae significantly reduced adult longevity, which could impact pest reproduction as adult flies require approximately two weeks to reach sexual maturity. Brief immersion of naturally infested oranges in 33–66 ppm spinosad solution also significantly reduced adult emergence. Exposure to spinosad-treated sand (33 ppm) did not adversely affect the foraging behavior or mortality of the braconid parasitoid Diachasmimorpha longicaudata. We conclude that effective control of A. ludens in soil with spinosad is possible but will likely require application of high concentrations of the insecticide, which may not be economically viable under conventional fruit production schemes. In the case of organic orchards surrounded by wild hosts that harbor large fly populations, targeted spinosad soil applications might be desirable as fly numbers could be significantly reduced without harming parasitoids.

The soil is the most important environmental reservoir for nucleopolyhedrovirus occlusion bodies (OBs). We employed a diet + soil bioassay technique to demonstrate that the earthworm Amynthas graci...

The inefficient production of nucleopolyhedrovirus occlusion bodies (OBs) can limit the commercialization of virus-based insecticides. The production of OBs in SfMNPV-infected Spodoptera frugiperda fourth instars was compared among groups of larvae that were frozen (−20 °C) immediately following death, or subjected to a 9-day period of post-mortem incubation at 5 °C or 15 °C. Incubation at 15 °C resulted in a ∼40% increase in total OB production and OBs per mg larval weight, compared to the −20 °C and 5 °C treatments. OBs from the 5 °C treatment were ∼10% smaller in cross sectional area and small OBs (

We study the first hitting time statistics between a one-dimensional run-and-tumble particle and a target site that switches intermittently between visible and invisible phases. The two-state dynamics of the target is independent of the motion of the particle, which can be absorbed by the target only in its visible phase. We obtain the mean first hitting time when the motion takes place in a finite domain with reflecting boundaries. Considering the turning rate of the particle as a tuning parameter, we find that ballistic motion represents the best strategy to minimize the mean first hitting time. However, the relative fluctuations of the first hitting time are large and exhibit non-monotonous behaviours with respect to the turning rate or the target transition rates. Paradoxically, these fluctuations can be the largest for targets that are visible most of the time, and not for those that are mostly invisible or rapidly transiting between the two states. On the infinite line, the classical asymptotic behaviour $\propto t^{-3/2}$ of the first hitting time distribution is typically preceded, due to target intermittency, by an intermediate scaling regime varying as $t^{-1/2}$. The extent of this transient regime becomes very long when the target is most of the time invisible, especially at low turning rates. In both finite and infinite geometries, we draw analogies with partial absorption problems., 5 figures

Amaranth (Amaranthus hypocondriacus L.) is a pseudocereal that is widely consumed in Mexico and other tropical regions, but which is attacked by Spodoptera exigua (Hubner) larvae. Products based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can be highly effective insecticides, but when applied to amaranth viral occlusion bodies (OBs) lost 50% of their activity in a 6 h period. A series of experiments was performed to examine the role of solar radiation, leaf oxidative enzymes, leaf pH and insect feeding behavior, all of which failed to explain the reduced prevalence of virus infection in S. exigua larvae on amaranth. The concentration of the flavonoid rutin on amaranth leaves was >200-fold higher than on another preferred host plant, sweet pepper (Capsicum annuum). However, in laboratory assays, rutin generally resulted in an increased prevalence of infection when administered in mixtures with semi-synthetic diet and OBs. We conclude that the cause of OB inactivation on amaranth remains elusive, although understanding how this will influence its efficacy as a biological insecticide requires field studies.

The effects of Poissonian resetting at a constant rate $r$ on the reaction time between a Brownian particle and a stochastically gated target are studied. The target switches between a reactive state and a non-reactive one. We calculate the mean time at which the particle subject to resetting hits the target for the first time, while the latter is in the reactive state. The search time is minimum at a resetting rate that depends on the target transition rates. When the target relaxation rate is much larger than both the resetting rate and the inverse diffusion time, the system becomes equivalent to a partially absorbing boundary problem. In other cases, however, the optimal resetting rate can be a non-monotonic function of the target rates, a feature not observed in partial absorption. We compute the relative fluctuations of the first hitting time around its mean and compare our results with the ungated case. The usual universal behavior of these fluctuations for resetting processes at their optimum breaks down due to the target internal dynamics., Comment: 5 figures

Resumen La hipercalcemia es un trastorno metabolico frecuente detectado en los analisis de bioquimica de rutina en pacientes hospitalizados. Sin embargo, en algunas ocasiones se comporta como un cuadro que amenaza la vida de los pacientes. Las etiologias mas frecuentes son el cancer y el hiperparatiroidismo primario. Los niveles sericos de calcio son regulados estrechamente, con valores normales de calcio total entre 2,2 y 2,6 mmol/l y niveles de calcio ionizado de 1,1 a 1,4 mmol/l. Pacientes con niveles de 2,6 a 3 mmol/l pueden ser asintomaticos, mientras que registros de pacientes en este rango o superiores determinan la aparicion de sintomas inespecificos que refleja el compromiso de multiples organos como son renal, gastrointestinal, neuromuscular, sistema nervioso central y ocasionalmente respiratorio. Este compromiso multiorganico condiciona una elevada mortalidad, la cual revierte usualmente despues de la cirugia con paratiroidectomia. Describimos el caso de una mujer adulta con disfuncion renal, respiratoria, cardiovascular, asociada a nefrolitiasis y pancreatitis aguda secundaria a hipercalcemia, producida por un adenoma paratiroideo que condiciona el desarrollo de panhipopituitarismo primario.

Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRas(G12D) and used (1)H (15)N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

The soil is the most important reservoir of baculovirus occlusion bodies (OBs) in the environment. The ability of the earthworm Eisenia fetida to transport OBs of Spodoptera frugiperda multiple nucleopolyhedrovirus was examined in laboratory terraria filled with an artificial soil. OBs were detected in soil samples using a soil-diet incorporation bioassay, for which the 50% lethal concentration was estimated at 2.7 × 10 6 OBs/g soil in S. frugiperda second instars. Incubation of earthworms in soil containing 10 9 OBs for 7 days did not result in a significant loss of OB virulence compared to soil without earthworms. The earthworm intestine was found to be slightly acidic, with acid-base indicators applied to lengths of dissected intestine suggesting a pH of 6.0–6.3. Despite their epigean habits, E. fetida individuals were observed to form burrows up to 22.5 cm deep in laboratory terraria. Soil-diet bioassays indicated the presence of OBs at depths of 6–8, 14–16 and 22–24 cm in samples taken at 1, 7 and 14 days following the application of 10 9 OBs to the surface of terraria containing earthworms. In contrast, OBs were only detected in samples from the soil surface in terraria without earthworms. We conclude that earthworms likely affect the distribution and dynamics of OB populations in soil habitats.

We study the non-equilibrium steady states and first passage properties of a Brownian particle with position $X$ subject to an external confining potential of the form $V(X)=\mu|X|$, and that is switched on and off stochastically. Applying the potential intermittently generates a physically realistic diffusion process with stochastic resetting toward the origin, a topic which has recently attracted a considerable interest in a variety of theoretical contexts but has remained challenging to implement in lab experiments. The present system exhibits rich features, not observed in previous resetting models. The mean time needed by a particle starting from the potential minimum to reach an absorbing target located at a certain distance can be minimized with respect to the switch-on and switch-off rates. The optimal rates undergo continuous or discontinuous transitions as the potential strength $\mu$ is varied across non-trivial values. A discontinuous transition with metastable behavior is also observed for the optimal strength at fixed rates., Comment: 7 Figures

Among the commitments undertaken by the governments participating in the OGP, there is a growing number of commitments to improve the provision of justice services. Successfully implementing these commitments can entail a reform opportunity that the justice sector needs so as to improve its legitimacy vis-a-vis society, as well as its internal functioning.

We will start this chapter by introducing the notion of innovation in the public sector and provide a brief overview on how this term has evolved, ultimately proposing four categories to classify public innovation (products, processes, governance and conceptual). We will then focus on innovation in justice as a new approach involving the different agencies and individuals that partake in and use justice-related services. We will use public innovation categories to classify different types of innovation in the justice sector and, to conclude, we will outline a few ideas which can lead to thinking about an open innovation model for the sector, focused on the interaction of justice institutions with civil society, other government institutions and the private sector.

In noisy environments such as the cell, many processes involve target sites that are often hidden or inactive, and thus not always available for reaction with diffusing entities. To understand reaction kinetics in these situations, we study the first hitting time statistics of a Brownian particle searching for a target site that switches stochastically between visible and hidden phases. At high crypticity, an unexpected rate limited power-law regime emerges for the first hitting time density, which markedly differs from the classic $t^{-3/2}$ scaling for steady targets. Our problem admits an asymptotic mapping onto a mixed, or Robin, boundary condition. Similar results are obtained with non-Markov targets and particles diffusing anomalously., Comment: 3 Figures

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

We study the dynamics of predator-prey systems where prey are confined to a single region of space and where predators move randomly according to a power-law (L\'evy) dispersal kernel. Site fidelity, an important feature of animal behaviour, is incorporated in the model through a stochastic resetting dynamics of the predators to the prey patch. We solve in the long time limit the rate equations of Lotka-Volterra type that describe the evolution of the two species densities. Fixing the demographic parameters and the L\'evy exponent, the total population of predators can be maximized for a certain value of the resetting rate. This optimal value achieves a compromise between over-exploitation and under-utilization of the habitat. Similarly, at fixed resetting rate, there exists a L\'evy exponent which is optimal regarding predator abundance. These findings are supported by 2D stochastic simulations and show that the combined effects of diffusion and resetting can broadly extend the region of species coexistence in ecosystems facing resources scarcity., Comment: 15 pages, 9 figures

The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the basis for viral insecticides in several countries in Latin America. The rate of acquisition of infection and the transmission of the pathogen from infected to healthy conspecifics both influence the efficacy of virus insecticides. By comparing the leaf area consumed by larvae with their stage-specific susceptibility to infection, we estimated that fourth instars were approximately twice as likely to acquire infection as second or third instars. Greenhouse trials indicated that 63% of third instars became infected within 1 h of exposure to soya plants treated with 1.5 × 107 occlusion bodies (OB)/m2 obtained from mixtures of local AgMNPV isolates, as did 90% of larvae sampled at 24 h. Acquisition of lethal infection was slower in field trials performed in the soya-growing Huastecas region of Mexico. Virus-induced mortality varied from 43 to 79% in natural infestations of larvae sampled at 72 h post-application depending on the dose of unformulated OBs applied with wetting agent, although the majority of larvae had already acquired a lethal infection at 24 h post-application. Spray application volume (200 or 400 l/ha) did not significantly affect the prevalence of infection at 1 h post-application. Infected fifth instars moved twice as far and died significantly higher on plants than control larvae. Overall, 22% of third instars that subsequently foraged on these plants acquired a lethal infection. We conclude that most insects acquire infection within 24 h of virus application and that OBs in feces and regurgitate of infected larvae contribute significantly to pathogen transmission., CDA received a student scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico.

[EN] In 1999, Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was introduced into a major soybean-growing region in Tamaulipas, Mexico, for control of its lepidopteran host, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae). The virus introduction proved to be highly successful in controlling this agronomically important pest. In order to determine the genotypic diversity and insecticidal traits of Mexican AgMNPVs, we obtained 30 field-collected isolates from Tamaulipas State. Five distinct variants (genotypes 1–5) were identified from plaques replicated in A. gemmatalis larvae by examination of restriction profiles using HindIII. Initial screening indicated that none of the variants, or co-occluded mixtures of variants in different proportions, was more pathogenic than the 30 field isolates mixture or a reference variant from Brazil (AgMNPV-2D). Mean occlusion body production also was similar among genotype variants, the mixture of 30 field isolates and AgMNPV-2D treatments, but was significantly reduced in 1 co-occluded mixture. Speed of kill also was similar among variants (except genotype 1) and their mixtures. Lethal concentration metrics indicated that these results were unlikely due to selection of variants with reduced pathogenicity during the plaque purification process. We conclude that the mixture of 30 field isolates most likely would prove suitable for use as a biological insecticide in the soybean-growing region of Mexico., [ES] En 1999, el nucleopoliedrovirus de Anticarsia gemmatalis (AgMNPV) fue introducido a una importante región de producción de soya en Tamaulipas, México para controlar el lepidóptero huésped, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae). La introducción del virus demostró ser muy exitosa en el control de esta importante plaga agrícola. Para determinar la diversidad genotípica y las características insecticidas de los AgMNPVs de México, obtuvimos 30 aislados colectados en campos de soya del estado de Tamaulipas. Mediante el uso de la enzima de restricción HindIII, se identificaron cinco variantes distintas (genotipos 1–5) que replicaron en A. gemmatalis. Los ensayos indicaron que ninguna de las variantes, ni mezclas coocluidas de las mismas en diferentes proporciones, tuvieron una patogenicidad mayor que la de la mezcla de 30 aislados silvestres, o de una variante de referencia de Brasil (AgMNPV-2D). La producción de cuerpos de oclusión fue similar entre las variantes, la mezcla de 30 aislados silvestres y el AgMNPV-2D, aunque la producción fue significativamente reducida en una mezcla de variantes coocluidas. El tiempo medio de matar también fue similar entre las variantes (excepto el genotipo 1) y las mezclas de variantes. Ensayos de concentración-mortalidad indicaron que durante el proceso de purificación de placa no hubo selección de variantes con menores características de patogenicidad. Concluimos que la mezcla de 30 aislados silvestres podría ser el ingrediente activo de un insecticida biológico en esta región de producción de soya en México., CDA received a doctoral scholarship from Mexico's Consejo Nacional de Ciencia y Tecnología and a mobility grant from the doctoral program of the Instituto de Ecología AC

The superfamily of cation/Ca(2+) exchangers includes both Na(+)/Ca(2+) exchangers (NCXs) and Na(+)/Ca(2+),K(+) exchangers (NCKX) as the families characterized in most detail. These Ca(2+) transporters have prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of the YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed, and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcina acetivorans. MaX1 catalyzes Ca(2+) uptake into membrane vesicles. The Ca(2+) uptake requires intravesicular Na(+) and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na(+)/Ca(2+) exchanger with kinetic properties similar to those of NCX. The availability of a prokaryotic Na(+)/Ca(2+) exchanger should facilitate structural and mechanistic investigations.

Spinosad is a naturally derived insecticide that has shown potential as a mosquito larvicide. To determine the activity of spinosad against blackflies, late-instar larvae from a community comprising Simulium triittatum (63.6%) and seven other species, including three known vectors of onchocerciasis in Mexico (S. metallicum, S. ochraceum, and S. callidum), were subjected to concentration-mortality laboratory bioassays following World Health Organization guidelines. Cephalic capsule measurements confirmed the relatively homogeneous distribution of experimental larvae. The 50% lethal concentration of spinosad was estimated at 1.48 ppm spinosad (95% confidence interval: 1.07-2.33) for a 10-min exposure period, whereas larvae treated with 0.05 ppm of the organophosphate temephos experienced 61% mortality. Immature aquatic insects were identified to genus and tested for their susceptibility to spinosad in the laboratory. After exposure to 12 ppm spinosad for 10 min, ephemeropterans, odonates, trichopterans, and hemipterans did not experience significantly increased mortality over that of untreated controls, whereas a significant increase in mortality was observed in spinosad-treated Plecoptera (P < 0.001). Tilapia and trout fry exposed to 12 ppm spinosad for 10 min did not experience increased mortality at 24-h postexposure over that of the controls. We conclude that spinosad is less toxic than temephos to these blackfly species, but is likely to have a low impact on nontarget members of the aquatic community.

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (Cin) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (Cout) to Cin ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted Cout topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with Cout topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

It has been previously established that nutrient pollution is the gravest and most prevalent problem of ocean health. Furthermore, upstream areas and non-point sources of pollution have been found to contribute with the majority of nutrients discharged into estuaries in the United States coastal zone. However, very scattered information is available related to upstream nutrient pollution sources with impact on the Mexican coast of the Gulf of Mexico. The objective of this paper is two-fold. One purpose is to review the information related to the impact of upstream sources of agro-industrial pollution which affect the Mexican coast of the Gulf of Mexico. The second purpose is to discuss the need of promoting cleaner production and environmentally sound biotechnologies as part of a strategy for the pollution prevention of such agro-industrial activities. The sugar industry was found to be by far the largest upstream nutrient pollution source. Coffee, alcohol and pig production were also found to be important pollution sources. A pollution prevention strategy based on water reuse and recycling, water reduction at the source and wastewater recycling by means of cost-effective and environmentally sound biotechnologies is proposed. Such a strategy could be part of the Integrated Coastal Area Management to be implemented for the states bordering the Gulf of Mexico.

An evaluation was made of the annual productivity of Spirulina (Arthrospira) and its ability to remove nutrients in outdoor raceways treating anaerobic effluents from pig wastewater under tropical conditions. The study was based at a pilot plant at La Mancha beach, State of Veracruz, Mexico. Batch or semi-continuous cultures were established at different seasons during four consecutive years. The protein content of the harvested biomass and the N and P removal from the ponds were also evaluated. Anaerobic effluents from digested pig waste were added in a proportion of 2% (v/v) to untreated sea-water diluted 1:4 with fresh water supplemented with 2 g L−1 sodium bicarbonate, at days 0, 3 and 5. A straight filament strain of Spirulina adapted to grow in this complex medium was utilized. A pH value 9.5 ± 0.2 was maintained. The productivity of batch cultures during summer 1998 was significantly more with a pond depth of 0.10 m than with a depth 0.065 m. The average productivity of semi-continuous cultures during summer 1999 was 14.4 g m−2 d−1 with a pond depth of 0.15 m and 15.1 g m−2 d−1 with a depth of 0.20 m. The average annual productivity for semi-continuous cultures operating with depths of 0.10 m for winter and 0.15 and 0.25 m for the rest of the year, was 11.8 g m−2 d−1. This is the highest value reported for a Spirulina cultivation system utilising sea-water. The average protein content of the semi-continuous cultures was 48.9% ash-free dry weight. NH4-N removal was in the range 84–96% and P removal in the range of 72–87%, depending on the depth of the culture and the season.

The Ras protein family consists of small GTPases that are critical signaling transducers regulating cellular processes including proliferation, differentiation, and survival. As monomeric GTPases, Ras proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Oncogenic mutations in one of the Ras isoforms, H-, N-, and K-Ras, impair GTP hydrolysis, thus de-regulating Ras signaling. Ras mutations are found in one-third of human cancers, and tumors bearing these mutations are notoriously difficult to treat. Conventional approaches to inhibit Ras with small molecules have been largely unsuccessful leading many to characterize the Ras family as an “undruggable” target. However, there is renewed enthusiasm towards Ras drug discovery in response to a recent surge of reports revealing structural complexities to Ras proteins that provide opportunity for innovative drug discovery efforts. In light of findings suggesting KRas contains transient and dynamic binding pockets, many of which are undetectable with traditional structural techniques, we used a second harmonic generation (SHG) platform to screen a ~2700 chemically diverse fragment collection for allosteric modulators of KRas. This high throughput SHG platform enables real-time measurement of protein conformational change under physiological conditions. We selected ~50 fragments for validation by SPR, and positive binders will be selected for structural and biochemical characterization. Another advantage of the SHG platform is that we can monitor the conformational dynamics of fully processed, farnesylated KRas bound to a phosphatidylserine-enriched membrane that mimics KRas’ physiological interaction with the plasma membrane. No other biophysical method is suitable to monitor the real-time interactions of small molecules or proteins with membrane-bound KRas. This analysis may reveal surface contacts unavailable in solution-based assays. We have successfully implemented SHG as a drug discovery platform against KRas, and we aim to utilize selected KRas conformational modulators towards the identification of novel binding pockets and towards the development of targeted KRas therapies. Citation Format: Elizabeth Donohue Vo, Gabriel Besserer Mercado, Patrick Alexander, Ben Moree, Que Van, Andrew G. Stephen, Joshua Salafsky, Frank McCormick. Identifying allosteric modulators of KRas using second harmonic generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2017-4018

Proteins populate a landscape of conformations that changes upon binding native ligands or drugs, altering protein function. Different conformations reveal distinct potential drug target sites, including allosteric sites distal to the active site of the protein. It has been historically difficult to identify allosteric modulators early in the screening process since the structural methods required to reveal binding sites are relatively low throughput.

The effect of two abundant, easily available and very low-cost agro-industrial organic residues, i.e., filter cake from the sugar industry and poultry litter, on the composting stabilization time of coffee pulp and on the quality of the produced compost, was evaluated. Piles of one cubic meter were built and monitored within the facilities of a coffee processing plant in the Coatepec region of the State of Veracruz, Mexico. Manual aeration was carried out once a week. A longer thermophilic period (28 days) and a much lower C/N ratio (in the range of 6.9–9.1) were observed in the piles containing the amendments, as compared to the control pile containing only coffee pulp (14 days and a C/N ratio of 14.4, respectively). The maximum assimilation rate of the reducing sugars was 1.6 g kg-1 d-1 (from 7.5 to 5.3%) during the first two weeks when accelerators were present in the proportion of 20% filter cake plus 20% poultry litter, while they accumulated at a rate of 1.2 g kg-1 d-1 (from 7.4 to 9.13%) during the same period in the control pile. The best combination of amendments was 30% filter cake with 20% poultry litter, resulting in a final nitrogen content as high as 4.81%. The second best combination was 20% filter cake with 10% poultry litter, resulting in a compost which also contained a high level of total nitrogen (4.54%). It was concluded that the use of these two residues enhanced the composting process of coffee pulp, promoting a shorter stabilization period and yielding a higher quality of compost.

Spatial and temporal regulation of intracellular Ca2+ concentrations is a fundamental requirement for life. The mammalian cardiac Na+-Ca2+ exchanger serves as the main mechanism for Ca2+ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca2+, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca2+. Together with mutational analysis of the Ca2+ binding sites, this study reveals the crucial role of one of the two bound Ca2+ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.

The use of untreated sea water supplemented with anaerobic effluents from digested pig waste and sodium bicarbonate was evaluated as a low-cost medium for semi-continuous cultivation of a mixed culture of two Spirulina strains in outdoor raceways under temperate climatic conditions (pond temperature in the range 21–26 °C and light intensity in the range 225–957␣μE m−2 s−1). The mixed culture had a predominant population (86.6 ± 3.9%) of an atypical Spirulina strain consisting of straight filaments, which appeared spontaneously after the strain with helicoidal trichomes had been subcultured. Morphological studies for the identification of the type and size of trichomes of the two strains (HF and SF) were carried out. The proportions of the two strains were observed to be stable during the monitoring period (30 days). Three different sets of semicontinuous cultures were carried out. Sets 1 and 2 were operated under regime 1 (a single addition of anaerobic effluents at time zero and no pH control) during the same season (June and July) of different years. Set 3 was operated under regime 2 (semi-continuous addition of anaerobic effluents and pH control) during the autumn. A minimum productivity of 3.6 g m−2 day−1 was obtained at one of the lowest temperatures (22.1 °C) and light intensities (245 μE m−2 s−1) and a maximum productivity of 10.9 g m−2 day−1 was observed at the highest temperature (25 °C) and highest average light intensity (618 μE m−2 s−1) registered for sets 1 and 2. The protein content in the Spirulina biomass harvested from these two sets varied from 17% to 65.6%. In set 3, a maximum productivity of 9.0 g m−2 day−1 was recorded at an average temperature of 24.4 °C and at an average light intensity of 668 μE m−2 s−1. The protein content in this set under regime 2 varied within a narrower range than in set 1 and set 2 (from 34.8% to 49.1%), apparently because of a continuous availability of ammonia nitrogen at a level of 30–50 mg l−1. However, in terms of the removal of ammonia nitrogen and chemical oxygen demand, regime 1 was more efficient than regime␣2.

Various technological options for recycling of sugar processing by-products and residuals are discussed making use of field data from five sugar factories in the State of Veracruz in Mexico and information from the literature. It is emphasized that diversification is urgently required in developing sugar exporting countries but that the feasibility of each technological option has to be assessed within each national and regional scenario. Bagasse is already recycled towards energy generation or sold to paper and pulp industries. It is recommended that the use of this by-product as a fuel should be optimied through various actions in such a way that a large saving of fossil fuel could be achieved. In the case of molasses, a strategy to increase ethanol production in the short, medium and long term is discussed. For the short term, the introduction of highly productive yeast strains and use of well trained personnel is recommended. For the medium and long term, development and adaptation of innovative technologies such as those involving continuous reactors and the use of Zymomonas mobilis are suggested. The strategy could be promoted through a National Programme aimed at increasing ethanol exportations, contributing to the benefit of the imports/exports balance. As stillage is the most important residual due to its high BOD and COD values, a discussion of at least five different alternative technologies for the recovery of the minerals or organic matter contained in it is presented. Such alternatives range from low to medium and high capital costs. Some of their advantages and disadvantages are discussed. Anaerobic digestion seems to be the best option whenever capital is available, although a secondary polishing treatment of the anaerobic effluents is required. In this case, an integrated system with recuperation of a multiproduct microalgae biomass (Spirulina sp.) appears to be a feasible option.

Gyrodactylids (Monogenea) are ectoparasites of fish, some of which negatively affect commercially valuable fishes. Temperature strongly regulates population dynamics of these viviparous flatworms in farmed and wild fish populations, with most gyrodactylid species showing positive temperature-abundance associations. In agreement with epidemiological theory, numerous laboratory studies demonstrate that these parasites cannot persist in confined fish populations without periodic introduction of susceptible hosts. Extinction of gyrodactylid populations is due to host immunity, which develops in several fish species. In this one-year study, we followed populations of the recognized pathogen Gyrodactylus cichlidarum infecting four genetic groups of confined tilapia (wild type Nile tilapia Oreochromis niloticus niloticus, red O. n. niloticus, Mozambique tilapia O. mossambicus and a red synthetic population called Pargo-UNAM) kept under farming conditions and subject to natural environmental fluctuations. Based on the antecedents given, we postulated the following three hypotheses: (1) parasite abundance will be regulated by water temperature; (2) parasites will induce host mortality, particularly during periods of rapid infrapopulation growth; and (3) gyrodactylid populations will eventually become extinct on confined fish hosts. We disproved the three hypotheses: (1) parasite numbers fluctuated independently of temperature but were associated to changes in microhabitat use; (2) although gyrodactylid populations exhibited considerable growth, no evidence was found of negative effects on the hosts; and (3) infections persisted for one year on confined fish. Microhabitat use changed over time, with most worms apparently migrating anteriorly from the caudal fin and ending on the pectoral fins. Gyrodactylid populations followed similar trajectories in all fish, aggregating and dispersing repeatedly. Several instances were found where increased parasite dispersion coincided with increased intensity of infection; as well as the opposite, where increased aggregation coincided with parasite population declines. Three alternative explanations could account for these observations: that parasites (1) experience differential mortality on different anatomical regions of the fish; (2) migrate to avoid intraspecific competition; and (3) migrate to escape localized immune responses induced by infection. Our data do not allow us to demonstrate which of these alternatives is correct, so we discuss the merits of each. We provide circumstantial evidence in support of the third explanation, because as shown in other fish host-gyrodactylid interactions where immune responses have been characterized, in this study worms progressively moved away from fins with high mucus cell density to those with low density - what would be anticipated if immune defenses occur and reach the fish surface through mucus.

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C(out) topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ∼3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

The Na + –Ca 2+ exchanger plays a central role in cardiac contractility by maintaining Ca 2+ homeostasis. Two Ca 2+ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca 2+ binding to these regulatory domains activates the transport of Ca 2+ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca 2+ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca 2+ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca 2+ ions in primary and secondary Ca 2+ sites. In the absence of Ca 2+ , Lys 585 stabilizes the structure by coordinating two acidic residues (Asp 552 and Glu 648 ), one from each of the Ca 2+ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca 2+ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca 2+ site completely remove Ca 2+ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na + –Ca 2+ exchanger.

Maximum protein accumulation (71%, w/w) and nutrient removal by a mutant strain of Spirulina maxima growing on sea water supplemented with anaerobically treated pig slurry was achieved at 30°C with constant illumination (60 to 70 μEm(-2)s(-1)), using a flow rate of 14.5 cm s(-1) (20 rev. min(-1) of a paddle wheel). Total phosphates were decreased by 99% and all ammonia-N was removed under these conditions.