Sporulation and Dispersal of the Biological Control Agent Aspergillus flavus AF36 Under Field Conditions.

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent.

Study of the competition between Colletotrichum godetiae and C. nymphaeae, two pathogenic species in olive.

Olive anthracnose, a critical olive fruit disease that adversely impacts oil quality, is caused by Colletotrichum species. A dominant Colletotrichum species and several secondary species have been identified in each olive-growing region. This study surveys the interspecific competition between C. godetiae, dominant in Spain, and C. nymphaeae, prevalent in Portugal, to shed light on the cause of this disparity. When Petri-dishes of Potato Dextrose Agar (PDA) and diluted PDA were co-inoculated with spore mixes produced by both species, C. godetiae displaced C. nymphaeae, even if the percentage of spores in the initial spore mix inoculation was just 5 and 95%, respectively. The C. godetiae and C. nymphaeae species showed similar fruit virulence in separate inoculations in both cultivars, the Portuguese cv. Galega Vulgar and the Spanish cv. Hojiblanca, and no cultivar specialization was observed. However, when olive fruits were co-inoculated, the C. godetiae species showed a higher competitive ability and partially displaced the C. nymphaeae species. Furthermore, both Colletotrichum species showed a similar leaf survival rate. Lastly, C. godetiae was more resistant to metallic copper than C. nymphaeae. The work developed here allows a deeper understanding of the competition between C. godetiae and C. nymphaeae, which could lead to developing strategies for more efficient disease risk assessment.

Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Resistance to Aspergillus flavus and Aspergillus parasiticus in Almond Advanced Selections and Cultivars and Its Interaction with the Aflatoxin Biocontrol Strategy.

Aflatoxin contamination of almond kernels, caused by Aspergillus flavus and A. parasiticus, is a severe concern for growers because of its high toxicity. In California, the global leader of almond production, aflatoxin can be managed by applying the biological control strain AF36 of A. flavus and selecting resistant cultivars. Here, we classified the almond genotypes by K-Means cluster analysis into three groups (susceptible [S], moderately susceptible [MS], or resistant [R]) based on aflatoxin content of inoculated kernels. The protective effects of the shell and seedcoat in preventing aflatoxin contamination were also examined. The presence of intact shells reduced aflatoxin contamination >100-fold. The seedcoat provided a layer of protection but not complete protection. In kernel inoculation assays, none of the studied almond genotypes showed a total resistance to the pathogen. However, nine traditional cultivars and four advanced selections were classified as R. Because these advanced selections contained germplasm derived from peach, we compared the kernel resistance of three peach cultivars to that shown by kernels of an R (Sonora) and an S (Carmel) almond cultivar and five pistachio cultivars. Overall, peach kernels were significantly more resistant to the pathogen than almond kernels, which were more resistant than pistachio kernels. Finally, we studied the combined effect of the cultivar resistance and the biocontrol strain AF36 in limiting aflatoxin contamination. For this, we coinoculated almond kernels of R Sonora and S Carmel with AF36 72 h before or 48 h after inoculating with an aflatoxin-producing strain of A. flavus. The percentage of aflatoxin reduction by AF36 strain was greater in kernels of Carmel (98%) than in those of Sonora (83%). Cultivar resistance also affected the kernel colonization by the biological control strain. AF36 strain limited aflatoxin contamination in almond kernels even when applied 48 h after the aflatoxin-producing strain. Our results show that biocontrol combined with the use of cultivars with resistance to aflatoxin contamination can result in a more robust protection strategy than the use of either practice in isolation.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

Divergent, stereoselective access to heterocyclic α,α-quaternary- and ß(2,3,3)-amino acid derivatives from a N-Pmp-protected Orn-derived ß-lactam.

A suitably protected Orn-derived (3S,4S)-ß-lactam was used as common intermediate in the synthesis of conformationally constrained (3S,4S)-2-oxoazepane α,α- and (2S,3S)-2-oxopiperidine-ß(2,3,3)-amino acid derivatives. Compared to alternative procedures using an N-p-methoxybenzyl group at the 2-azetidinone, the incorporation of a p-methoxyphenyl moiety is crucial for the excellent stereochemical outcomes in the preparation of these heterocyclic amino acids. Chemoselective 7- or 6-exo-trig cyclization was achieved through alternative sequences of Pmp-deprotection/Boc-activation, followed by inter- and intramolecular ß-lactam ring opening, respectively.

Experimental and theoretical studies on the rearrangement of 2-oxoazepane α,α-amino acids into 2'-oxopiperidine ß(2,3,3) -amino acids: an example of intramolecular catalysis.

Enantiopure ß-amino acids represent interesting scaffolds for peptidomimetics, foldamers and bioactive compounds. However, the synthesis of highly substituted analogues is still a major challenge. Herein, we describe the spontaneous rearrangement of 4-carboxy-2-oxoazepane α,α-amino acids to lead to 2'-oxopiperidine-containing ß(2,3,3) -amino acids, upon basic or acid hydrolysis of the 2-oxoazepane α,α-amino acid ester. Under acidic conditions, a totally stereoselective synthetic route has been developed. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six-membered heterocycle. A quantum mechanical study was carried out to obtain insight into the remarkable ease of this rearrangement, which occurs at room temperature, either in solution or upon storage of the 4-carboxylic acid substituted 2-oxoazepane derivatives. This theoretical study suggests that the rearrangement process occurs through a concerted mechanism, in which the energy of the transition states can be lowered by the participation of a catalytic water molecule. Interestingly, it also suggested a role for the carboxylic acid at position 4 of the 2-oxoazepane ring, which facilitates this rearrangement, participating directly in the intramolecular catalysis.

Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Exploring the Phe-Gly dipeptide-derived piperazinone scaffold in the search for antagonists of the thrombin receptor PAR1.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Highly functionalized 2-oxopiperazine-based peptidomimetics: an approach to PAR1 antagonists.

A series of pseudodipeptide-based chiral 1,3,4,5-tetrasubstituted-2-oxopiperazines has been designed and synthesized as potential PAR1 antagonists. These highly functionalized piperazines were synthesized from aromatic and basic amino acid derived Ψ[CH(CN)NH]pseudodipeptides through a four step pathway that involves reduction of the cyano group to build the 2-oxopiperazine ring, followed by selective functionalization at the N4-, N1-positions, and at the exocyclic moiety at position C5. This regioselective functionalization required the fine tuning of reaction conditions. All new compounds were screened as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cell lines. Some of the compounds displayed moderate PAR1 antagonist activity, while, others were cytotoxic at µM concentration. No correlation was observed between both types of activities.

Solvent-free synthesis of α-amino nitrile-derived ureas.

An efficient and environmentally friendly methodology for the solvent-free synthesis of α-amino nitrile derived ureas from α-amino acid based amino nitriles has been developed. At room temperature no epimerization was observed in the resulting ureas, but under microwave heating, epimerization occurred at the chiral center bearing the cyano group.

Highly functionalized 1,2-diamino compounds through reductive amination of amino acid-derived ß-keto esters.

1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived ß-keto esters are a suitable starting point for the synthesis of ß,γ-diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α-amino esters. AcOH and NaBH(3)CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the ß,γ-diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity.

Eficacia de la estimulación cognitiva en pacientes con enfermedad de Alzheimer / Cognitive stimulation efficacy in patients with Alzheimer's disease

En la actualidad, son cada vez más los profesionales que se decantan por la intervención en la enfermedad de Alzheimer (EA) con un tratamiento combinado, y la estimulación cognitiva (EC) obtiene paulatinamente un papel más relevante. El objetivo de este estudio fue comprobar que la EC muestra resultados de mantenimiento de las funciones cognitivas frente a los sujetos que no reciben este tratamiento. Pacientes y método: Participaron un total de 52 sujetos diagnosticados de EA y con Mini-Mental State Examination (MMSE) >=20, divididos en dos grupos: un grupo experimental compuesto por 31 sujetos que estuvieron expuestos a sesiones de EC durante 9 meses, y un grupo control formado por 21 pacientes que no realizaron EC. La EC se llevó a cabo en la Clínica Neurodem por al menos una neuropsicóloga en sesiones grupales de 6 personas como máximo, 2 veces a la semana y con una duración de 120 minutos por sesión, donde se trabajaron diferentes funciones cognitivas utilizando distintas técnicas. Resultados: Los que obtuvimos fueron una media de MMSE del grupo experimental antes de EC de 24,61 con una DT de 2,58, y una media después de EC de 24,61 con DT de 3,73. En el grupo control encontramos una media inicial de MMSE de 22,80 y DT de 2,27, y después de 9 meses una media de MMSE de 21,42 y DT de 1,71. Las puntuaciones en fluencia fueron, para el grupo experimental, en pretest de 13 y en postest de 13,18; para los controles, la media en fluencia fue de 10,61 y, tras 9 meses, de 8,57. Conclusiones: El grupo experimental mantiene estables sus puntaciones en ambas pruebas frente al grupo control, que tiene un descenso en ellas(AU)

Nowadays, there are more professionals who choose intervention in Alzheimer Disease (AD) with a combined treatment, this way, cognitive stimulation (CS) becomes more relevant day by day. The aim of this study was to prove that CS shows maintenance’s results of the cognitive functions in patients who received this treatment versus patients that did not recieved it. Patients and method: 52 subjects with AD and Mini-Mental State Examination (MMSE) >=20, divided in two groups: experimental group (n= 31) which executed CS during 9 months, and one control group (n= 21) which didn’t execute the task. The CS was developed in Neurodem by one of the neuropsychologist which works in the clinic. It was taken in grupal session with a maximum of 6 patients, twice a week during 120 minutes, where the neuropsychologist and the patients worked with different cognitive functions, using different tecnics. Results: before CS experimental group x MMSE was 24,61 and sigma = 2,58 and control group MMSE x = 22,80 and sigma= 2,27. After 9 months results shows that x MMSE in experimental group was 24,61 and sigma was 2,58. In control group MMSE x was 21,42 and sigma was 1,71. In experimental group fluency puntuation was 10,61 and before 9 months was 8,57. Conclusions: the experimental group keeps its scores in both tasks whereas the control group shows a reduction in these tasks(AU)

Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1.

By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity.

Azepane quaternary amino acids as effective inducers of 3(10) helix conformations.

A simple method for the synthesis of an azepane quaternary amino acid in enantiopure form is described. Theoretical, NMR, and X-ray studies indicated that this azepane-derived amino acid is an effective stabilizer of 3(10) helical structures in short peptides.

Cyclic amino acid linkers stabilizing key loops of brain derived neurotrophic factor.

Based on ß-turn-like BDNF loops 2 and 4, involved in receptor interaction, cyclic peptide replicas were designed, synthesized and tested. In addition to the native turn residues, the cyclic peptides include a linker unit between the N- and C-termini, selected by molecular modeling among various non-proteinogenic cyclic amino acids. NMR conformational studies showed that most of the cyclic peptides were able to adopt turn-like structures. Several of the analogues displayed significant inhibition of the BDNF-induced TrkB receptor phosphorylation, and hence could be useful templates for developing improved antagonists for this receptor.

Optically active 1,3,4,4-tetrasubstituted ß-lactams: synthesis and evaluation as tumor cell growth inhibitors.

The in vitro cytotoxicity assays of several enantiopure (3S,4S)- and (3R,4R)-1,3,4,4-tetrasubstituted ß-lactams derived from amino acids have shown that the (3S,4S)-4-benzyl-1-p-methoxybenzyl-3-methyl-4-methoxycarbonyl derivative 2a, obtained from Phe, displays significant activity, which is comparable to that of the anticancer drug Doxorubicin against HT29 cell lines. Modifications at positions 1 and 4 of the ß-lactam ring led to identify the Tyr(2,6-ClBz) analogu 26d with similar activity data to those of 2a. The synthesis and SAR of all these tetrasubstituted ß-lactams are reported here.

Quaternary α,α-2-oxoazepane α-amino acids: synthesis from ornithine-derived ß-lactams and incorporation into model dipeptides.

To explore further the chemistry of amino acid-derived ß-lactams, their conversion to α,α-heterocyclic quaternary amino acid derivatives is investigated. The latter derivatives, containing 2-oxoazepane as the α,α-substituent, are synthesized by a simple Pd-C-catalyzed hydrogenolysis of Orn(Z)-derived 2-azetidinones. The rearrangement from four- to seven-membered lactam ring is driven by the key intramolecular opening of the 1-Boc-ß-lactam, initiated by 7-exotrig ring closure from the NH(2) of the Orn side chain. The synthetic route is applied to the stereoselective preparation of enantiomerically pure 4-amino-3-methyl-2-oxoazepane-4-carboxylate derivatives, for which the structure and configuration is confirmed by X-ray diffraction. Molecular modeling and NMR experiments indicate that these quaternary amino acids are able to drive the adoption of ß-turn secondary structures when incorporated in model dipeptide derivatives.

TRPV1 modulators: structure-activity relationships using a rational combinatorial approach.

A discrete library of linear and hydantoin-containing dipeptide derivatives, based on the Lys-Trp(Nps) scaffold, was prepared by solid-phase synthesis. SAR studies indicated that potency for TRPV1 blockade and selectivity towards NMDA is mainly dictated by the side-chain length and the basic nature of α, ω-groups in the N-terminal residue. The 2-Nps moiety at position 2 of Trp indole ring is preferred over the 2-pyridine one.