Search

Cationic ultrashort lipopeptides (USLPs) are promising antimicrobial candidates to combat multidrug-resistant bacteria. Using DICAMs, a newly synthesized family of tripeptides with net charges from −2 to +1 and a fatty amine conjugated to the C-terminus, we demonstrate that anionic and neutral zwitterionic USLPs can possess potent antimicrobial and membrane-disrupting activities against prevalent human pathogens such as Streptococcus pneumoniae and Streptococcus pyogenes. The strongest antimicrobials completely halt bacterial growth at low micromolar concentrations, reduce bacterial survival by several orders of magnitude, and may kill planktonic cells and biofilms. All of them comprise either an anionic or neutral zwitterionic peptide attached to a long fatty amine (16–18 carbon atoms) and show a preference for anionic lipid membranes enriched in phosphatidylglycerol (PG), which excludes electrostatic interactions as the main driving force for DICAM action. Hence, the hydrophobic contacts provided by the long aliphatic chains of their fatty amines are needed for DICAM’s membrane insertion, while negative-charge shielding by salt counterions would reduce electrostatic repulsions. Additionally, we show that other components of the bacterial envelope, including the capsular polysaccharide, can influence the microbicidal activity of DICAMs. Several promising candidates with good-to-tolerable therapeutic ratios are identified as potential agents against S. pneumoniae and S. pyogenes. Structural characteristics that determine the preference for a specific pathogen or decrease DICAM toxicity have also been investigated.

Intermittent rivers and ephemeral streams (IRES) compose the majority of draining networks on Earth, supporting a unique fraction of biodiversity. Despite their high ecological value, IRES are increasingly threatened by global change and require appropriate biomonitoring and restoration tools. However, indices and indicators used in routine biomonitoring programs are often confounded by drying effects. This occurs because most pollution-sensitive taxa are lost over drying gradients, limiting the utility of current biomonitoring tools in IRES. To address this challenge, there is a need to evaluate which taxa should be used to calculate biomonitoring metrics and indicators over the different portions of the drying gradient. Here, using high-resolution drying data from 33 unpolluted streams, we explored the drying preferences of macroinvertebrates to identify their potential as biomonitoring indicators in IRES. To do this, we characterized macroinvertebrate drying niches and identified those with drying resistance and sensitivity preferences. Next, we evaluated the capacity of functional traits to predict drying specialization and affinity. Finally, to identify potential biomonitoring metrics and indicators for IRES, we evaluated how drying influenced the density, relative abundance and richness of taxa in drying-sensitive and drying-resistant niches. Our results identified three macroinvertebrate groups with drying-resistant niches (partly tolerant, generalist and specialist taxa) and one group of drying-sensitive taxa. We also found that functional traits had a limited capacity to represent differences in drying niches, with shredding trophic preferences and body size showing the strongest correlations. In addition, we observed that the density, relative abundance and richness of drying-resistant taxa were less influenced by drying intensity than those of drying-sensitive taxa. Finally, we found that some pollution-sensitive taxa with partial (e.g., Lepidostoma), moderate (e.g., Corduliidae) or high drying tolerance (e.g., Nemoura) can serve as potential indicators in IRES. Taken together, our results demonstrate that characterizing drying niches can be a useful strategy for developing biomonitoring tools in IRES and for highlighting the limitations of taxonomic and trait-based approaches.

Abstract Background Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

ABSTRACT Expediting drug discovery to fight antibacterial resistance requires holistic approaches at system levels. In this study, we focused on the human-adapted pathogen Haemophilus influenzae, and by constructing a high-quality genome-scale metabolic model, we rationally identified new metabolic drug targets in this organism. Contextualization of available gene essentiality data within in silico predictions identified most genes involved in lipid metabolism as promising targets. We focused on the β-ketoacyl-acyl carrier protein synthase III FabH, responsible for catalyzing the first step in the FASII fatty acid synthesis pathway and feedback inhibition. Docking studies provided a plausible three-dimensional model of FabH in complex with the synthetic inhibitor 1-(5-(2-fluoro-5-(hydroxymethyl)phenyl)pyridin-2-yl)piperidine-4-acetic acid (FabHi). Validating our in silico predictions, FabHi reduced H. influenzae viability in a dose- and strain-dependent manner, and this inhibitory effect was independent of fabH gene expression levels. fabH allelic variation was observed among H. influenzae clinical isolates. Many of these polymorphisms, relevant for stabilization of the dimeric active form of FabH and/or activity, may modulate the inhibitory effect as part of a complex multifactorial process with the overall metabolic context emerging as a key factor tuning FabHi activity. Synergies with antibiotics were not observed and bacteria were not prone to develop resistance. Inhibitor administration during H. influenzae infection on a zebrafish septicemia infection model cleared bacteria without signs of host toxicity. Overall, we highlight the potential of H. influenzae metabolism as a source of drug targets, metabolic models as target-screening tools, and FASII targeting suitability to counteract this bacterial infection. IMPORTANCE Antimicrobial resistance drives the need of synergistically combined powerful computational tools and experimental work to accelerate target identification and drug development. Here, we present a high-quality metabolic model of H. influenzae and show its usefulness both as a computational framework for large experimental data set contextualization and as a tool to discover condition-independent drug targets. We focus on β-ketoacyl-acyl carrier protein synthase III FabH chemical inhibition by using a synthetic molecule with good synthetic and antimicrobial profiles that specifically binds to the active site. The mechanistic complexity of FabH inhibition may go beyond allelic variation, and the strain-dependent effect of the inhibitor tested supports the impact of metabolic context as a key factor driving bacterial cell behavior. Therefore, this study highlights the systematic metabolic evaluation of individual strains through computational frameworks to identify secondary metabolic hubs modulating drug response, which will facilitate establishing synergistic and/or more precise and robust antibacterial treatments.

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3β6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.

Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression.

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.

Bacterial surfaces are decorated with distinct carbohydrate structures that may substantially differ among species and strains. These structures can be recognized by a variety of glycan-binding proteins, playing an important role in the bacteria cross-talk with the host and invading bacteriophages, and also in the formation of bacterial microcolonies and biofilms. In recent years, different microarray approaches for exploring bacterial surface glycans and their recognition by proteins have been developed. A main advantage of the microarray format is the inherent miniaturization of the method, which allows sensitive and high-throughput analyses with very small amounts of sample. Antibody and lectin microarrays have been used for examining bacterial glycosignatures, enabling bacteria identification and differentiation among strains. In addition, microarrays incorporating bacterial carbohydrate structures have served to evaluate their recognition by diverse host/phage/bacterial glycan-binding proteins, such as lectins, effectors of the immune system, or bacterial and phagic cell wall lysins, and to identify antigenic determinants for vaccine development. The list of samples printed in the arrays includes polysaccharides, lipopoly/lipooligosaccharides, (lipo)teichoic acids, and peptidoglycans, as well as sequence-defined oligosaccharide fragments. Moreover, microarrays of cell wall fragments and entire bacterial cells have been developed, which also allow to study bacterial glycosylation patterns. In this review, examples of the different microarray platforms and applications are presented with a view to give the current state-of-the-art and future prospects in this field.

Se realizó un estudio descriptivo, retrospectivo y transversal de 87 pacientes con tumores cutáneos no melanoma, atendidos en el Servicio de Dermatología del Hospital Clinicoquirúrgico DocenteDr. Joaquín Castillo Duany de Santiago de Cuba, desde enero de 2012 hasta igual mes de 2014 con el objetivo de caracterizarles según variables clínicas y epidemiológicas de interés para la investigación. La información se recogió de las historias clínicas y de los informes de anatomía patológica. Como medidas de resumen para variables cuantitativas y cualitativas se utilizaron la media, la frecuencia absoluta y la relativa. En la serie predominaron el carcinoma basocelular (60,0 %), el sexo masculino, los pacientes de piel blanca y los mayores de 50 años de edad; asimismo, la cabeza resultó ser la región topográfica más afectada y la forma clínica nodular la más frecuente. Según la ocupación, los agricultores y los albañiles estuvieron mayormente relacionados con este tipo de cáncer de piel.

The study of leaf litter as a resource for shredders has emerged as a key topic in trophic links in ecology. However, thus far, most studies have emphasized the leaf quality as one of the main determinants of shredder behaviour and growth without simultaneously considering the leaf quantity availability. Nevertheless, the combined effects of leaf quantity and quality on shredder behaviour and growth is particularly crucial to further understand how ecosystem functioning may respond to the increasing flow intermittency due to climate change. In this study, we explore how changes in the leaf litter quality and quantity influence the feeding preferences and growth of an invertebrate shredder (Potamophylax latipennis). To do so, we used black poplar leaves conditioned in two streams with different flow regimens as a food resource. Afterwards, using a microcosm approach, we offered leaf discs that varied in terms of leaf quantity and quality to P. latipennis. Our results showed that flow intermittency had a negative effect on the quality of the food resource, and a lower quality had a negative effect on the consumption and growth rates of P. latipennis. Furthermore, we found that P. latipennis fed selectively on higher quality leaves even though the availability (quantity) of this resource was lower. In the context of climate change, with higher aridity/drier conditions/scenarios, our findings suggest that a decrease in the availability (quantity) of high-quality resources could potentially threaten links in global fluvial food webs and thus threaten ecosystem functioning.

Initiation of plasmid rolling circle replication is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed.

The emergence and spread of antibiotic-resistant bacteria is pushing the need of alternative treatments. In this context, phage therapy is already a reality to successfully fight certain multiresistant bacteria. Among different phage gene products, murein hydrolases responsible of phage progeny liberation (also called lysins or endolysins) are weapons that target specific peptidoglycan bonds, leading to lysis and death of susceptible bacteria when added from the outside. In the pneumococcal system, all but one phage murein hydrolases reported to date share a choline-binding domain that recognizes cell walls containing choline residues in the (lipo)teichoic acids. Some purified pneumococcal or phage murein hydrolases, as well as several chimeric proteins combining natural catalytic and cell wall-binding domains have been used as effective antimicrobials. In this work we have constructed a novel chimeric N-acetylmuramoyl-L-alanine amidase (PL3) by fusing the catalytic domain of the Pal amidase (a phage-coded endolysin) to the cell wall-binding domain of the LytA amidase, the major pneumococcal autolysin. The physicochemical properties of PL3 and the bacteriolytic effect against several pneumococci (including 48 multiresistant representative strain) and related species, like Streptococcus pseudopneumoniae, Streptococcus mitis and Streptococcus oralis, have been studied. Results have shown that low doses of PL3, in the range of 0.5–5 µg/ml, are enough to practically sterilize all choline-containing strains tested. Moreover, a single 20-µg dose of PL3 fully protected zebrafish embryos from infection by S. pneumoniae D39 strain. Importantly, PL3 keeps 95% enzymatic activity after four weeks at 37ºC and can be lyophilized without losing activity, demonstrating a remarkable robustness. Such stability, together with a prominent efficacy against a narrow spectrum of human pathogens, confers to PL3 the characteristic to be an effective therapeutic. In addition, our results demonstrate that the structure/function-based domain shuffling approach is a successful method to construct tailor-made endolysins with higher bactericidal activities than their parental enzymes.

Se presenta el caso clínico de un recién nacido a término por parto distócico (cesárea), debido a una toxemia gravídica, con antecedente de polihidramnios, que manifestó dificultad respiratoria a los pocos minutos del nacimiento y fue ingresado en el Hospital Infantil Norte "Dr. Juan de la Cruz Martínez Maceira" de Santiago de Cuba. Le fueron detectadas cifras de glucemia muy bajas que, evolutivamente, trajeron aparejadas convulsiones tónico-clónicas generalizadas, cuyas frecuencias no se lograban disminuir. Se le diagnosticó una hipoglucemia hiperinsulínica persistente (nesidioblastosis) y fue trasladado a La Habana para recibir tratamientos clínico y quirúrgico definitivos. Actualmente es atendido por un equipo médico multidisciplinario.The case of a term infant by dystocia (cesarean section) due to pregnancy toxemia with history of polyhydramnios is presented, who had respiratory distress a few minutes after birth and he was admitted to "Dr. Juan de la Cruz Martínez Maceira" Northern Children Hospital of Santiago de Cuba. Very low blood glucose levels were detected that progressively caused tonic-clonic seizures, which frequencies could not be reduced. He was diagnosed with persistent hyperinsulinemic hypoglycemia (nesidioblastosis) and was transferred to Havana to receive definitive clinical and surgical treatments. Currently, he is treated by a multidisciplinary medical team.

Se presenta el caso clínico de un infante de 4 años de edad, con antecedente de amigdalitis a repetición, ingresado en el Hospital Infantil Norte Docente "Dr. Juan de la Cruz Martínez Maceira" de Santiago de Cuba por presentar, desde hacía 9 días, fiebre de 38 y 39 °C, odinofagia, aumento de volumen del cuello y erupción cutánea generalizada. Teniendo en cuenta el examen físico y los resultados de los estudios complementarios efectuados se diagnosticó la enfermedad de Kawasaki, por lo cual se indicó tratamiento con Intacglobin®, Aspirina® y vitaminoterapia. El paciente evolucionó favorablemente y egresó de la institución hospitalaria sin complicaciones.The case of a 4 year-old child with history of recurrent tonsillitis, who was admitted to "Dr. Juan de la Cruz Martínez Maceira" Northern Teaching Children Hospital of Santiago de Cuba is reported for presenting with temperature of 38 and 39°C, odynophagia, enlargement of the neck and generalized rash in the past nine days. Based on physical examination and the results of complementary tests the Kawasaki disease was diagnosed, for which a treatment with Intacglobin®, Aspirin®, and vitamin therapy was indicated. The patient made good progress and was discharged from the hospital without complications.

Se realizó un estudio descriptivo y transversal de los 1 311 pacientes que recibieron antimicrobianos de amplio espectro en el Hospital Infantil Norte Docente "Dr. Juan de la Cruz Martínez Maceira" de Santiago de Cuba, desde enero hasta diciembre de 2009, a fin de analizar el uso de estos medicamentos, para lo cual se revisaron las historias clínicas correspondientes y los controles del Departamento de Farmacia. En la casuística predominaron la ceftriaxona como medicamento más empleado (64,8 %), la Unidad de Cuidados Intensivos como el servicio que más lo utilizó (41,7 %), la neumonía bacteriana como entidad clínica más común (42,0 %) y la evolución favorable de los afectados (98,5 %).A descriptive and cross-sectional study of the 1 311 patients who received wide spectrum antimicrobials in "Dr. Juan de la Cruz Martínez Maceira" Teaching Northern Children Hospital in Santiago de Cuba was carried out from January to December, 2009, in order to analyze the use of these drugs. For this aim, the corresponding medical records and the controls of the Pharmacy Department were reviewed. Ceftriaxone as the most used medication (64.8 %), the Intensive Care Unit as the most used service (41.7 %), the bacterial pneumonia as the most common clinical entity (42.0 %) and the favorable clinical course of those affected (98.5 %) prevailed in the case material.

Se realizó un estudio descriptivo y longitudinal de 105 menores de 18 años con neumonía bacteriana grave, ingresados en el Hospital Infantil Norte Docente "Dr. Juan de la Cruz Martínez Maceira" de Santiago de Cuba durante el 2010, con vistas a caracterizarles según algunas variables clínico epidemiológicas de interés para la investigación. En la casuística predominaron el sexo masculino (54,0 %), las edades de 1 a 6 meses (32,0 %), la infección extra hospitalaria (89,0 %) y la procedencia del municipio de Songo-La Maya (21,0 %). Los pacientes fueron hospitalizados básicamente en estado grave (74, 2 %), sobre todo a causa de 2 factores de riesgo: desnutrición y urbanización (57,0 y 51,0 %, respectivamente).A descriptive and longitudinal study of 105 children under 18 years with severe bacterial pneumonia, admitted to "Dr. Juan of the Cruz Martínez Maceira" Teaching Northern Children Hospital from Santiago de Cuba was carried out during the 2010, with the objective of characterizing them according to some clinical and epidemiological variables of interest for the investigation. Male sex (54,0 %), ages from 1 to 6 months (32,0 %), community acquired infection (89,0 %) and the origin from Songo-La Maya municipality (21,0 %) prevailed in the case material. The patients were admitted basically in severe condition (74, 2 %), mainly because of 2 risk factors: malnutrition and urbanization (57,0 and 51,0 %, respectively).

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

This work aims to characterise the current autotrophic compartment of the Albufera des Grau coastal lagoon (Menorca, Balearic Islands) and to assess the relationship between the submerged macrophytes and the limnological parameters of the lagoon. During the study period the submerged vegetation was dominated by the macrophyte Ruppia cirrhosa, which formed dense extensive meadows covering 79% of the surface. Another macrophyte species, Potamogeton pectinatus, was also observed but only forming small stands near the rushing streams. Macroalgae were only occasionally observed. Macrophyte biomass showed a clear seasonal trend, with maximum values in July. The biomass of R. cirrhosa achieved 1760 g DW m-2, the highest biomass ever reported for this species in the literature. The seasonal production-decomposition cycle of the macrophyte meadows appears to drive the nutrient dynamics and carbon fluxes in the lagoon. Despite the significant biomass accumulation and the absence of a washout of nutrients and organic matter to the sea, the lagoon did not experience a dystrophic collapse. These results indicate that internal metabolism is more important than exchange processes in the lagoon.

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.

The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

The effect of dissolved nutrients on growth, nutrient content and uptake rates of Chaetomorpha linum in a Mediterranean coastal lagoon (Tancada, Ebro delta, NE Spain) was studied in laboratory experiments. Water was enriched with distinct forms of nitrogen, such as nitrate or ammonium and phosphorus. Enrichment with N, P or with both nutrients resulted in a significant increase in the tissue content of these nutrients. N-enrichment was followed by an increase in chlorophyll content after 4 days of treatment, although the difference was only significant when nitrate was added without P. P-enrichment had no significant effect on chlorophyll content. In all the treatments an increase in biomass was obseved after 10 days. This increase was higher in the N+P treatments. In all the treatments the uptake rate was significantly higher when nutrients were added than in control jars. The uptake rate of N, as ammonium, and P were significantly higher when they were added alone while that of N as nitrate was higher in the N+P treatment. In the P-enriched cultures, the final P-content of macroalgal tissues was ten-fold that of the initial tissue concentrations, thereby indicating luxury P-uptake. Moreover, at the end of the incubation the N:P ratio increased to 80, showing that P rather than N was the limiting factor for C. linum in the Tancada lagoon. The relatively high availability of N is related to the N inputs from rice fields that surround the lagoon and to P binding in sediments.

Ceragenin CSA-13, a cationic steroid, is here reported to show a concentration-dependent bactericidal/bacteriolytic activity against pathogenic streptococci, including multidrug-resistant Streptococcus pneumoniae. The autolysis promoted by CSA-13 in pneumococcal cultures appears to be due to the triggering of the major S. pneumoniae autolysin LytA, an N-acetylmuramoyl-L-alanine amidase. CSA-13 also disintegrated pneumococcal biofilms in a very efficient manner, although at concentrations slightly higher than those required for bactericidal activity on planktonic bacteria. CSA-13 has little hemolytic activity which should allow testing its antibacterial efficacy in animal models.

Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0-6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-l-Ala-d-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities.

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these "neurotoxic proteins" triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases.

In intermittent streams, aquatic organisms use various strategies to face dry phases, but the relative contribution of these strategies to persist during dry phase remains unclear. Here, we investigated the in situ persistence of benthic invertebrates in the saturated hyporheic sediments and the “invertebrate seedbank” that persists in dry sediments across six Mediterranean intermittent streams. Taxonomic and functional responses within hyporheic and seedbank assemblages were compared with those in the benthic assemblages under connected flow conditions by combining field and mesocosms data. The dry phase duration in each stream was calculated to assess the responses of hyporheic and seedbank assemblages. Taxonomic composition and abundance-weighted traits related to resistance and resilience to face the dry phase in each assemblage type (benthic, hyporheic, seedbank) were determined. Taxonomic (richness and diversity) and functional (richness and dispersion) metrics were also calculated. We found that seedbank and hyporheic assemblages supported up to 16% and 40% of the benthic taxa, respectively. Only taxonomic and functional richness differed between assemblage types. Contrary to previous research, no clear relationship was established between diversity or the abundance of resistance traits and the duration of dry phase; however, a negative linear relationship was identified between the abundance of resilience traits and the duration of the dry phase. The increase in the frequency and duration of drying events due to climate change will reduce water availability in both saturated and unsaturated streambed sediments, compromising the persistence of aquatic biodiversity in intermittent streams. [ABSTRACT FROM AUTHOR]

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.