The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants.

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

Transcriptomics and interactomics during the primary infection of an SfNPV baculovirus on Spodoptera frugiperda larvae.

The fall armyworm (FAW), Spodoptera frugiperda, has been the most devastating pest of corn as well as of other crops in America, and more recently in Africa and Asia. The development of resistance to chemical insecticides led the search for environmentally friendly biological alternatives such as baculoviruses. This study focuses on the primary infection of the baculovirus SfNPV-Ar in the FAW's midgut epithelium, by analyzing the differential expression of transcripts in excised midguts at 6, 12, and 24 h post-infection (hpi), and predicted their interactions. Interaction of viral factors with the infected midgut tissue could alters various cellular processes, such as the apoptotic system due to the up-regulation observed of FABP at 6 hpi and of HSP90 at 24 hpi, along with the down-regulated PRX at 6 hpi and FABP transcripts between 12 and 24 hpi. Changes in transcript regulation could affect the cellular architecture of infected cells due to up-regulation of ARP 2/3 at 6 and 12 hpi, followed by down-regulation at 24 hpi. In relation to protein folding proteins, HSP90 was up-regulated at 24 hpi and PDI was down-regulated between 6 and 12 hpi. With respect to metabolism and cellular transport, AcilBP and ATPS0 were up regulated at 6 hpi and 12 hpi, respectively. In reference to transcription and translation up-regulation of RPL11 at 6 hpi and of FPN32 and RPL19 at 24 hpi was detected, as well as the down-regulation of RPL19 at 6 hpi, of PDI and RPL7 at 12 hpi, and of FABP at 24 hpi. In conclusion, gene regulation induced by viral infection could be related to the cytoskeleton and cellular metabolism as well as to oxidative stress, apoptosis, protein folding, translation, and ribosomal structure. The results presented in this work are an approach to understanding how the virus takes control of the general metabolism of the insect host during the primary infection period.

Contaminants of emerging concern fate and fluvial biofilm status as pollution markers in an urban river.

Contaminants of emerging concern (CEC) are still under research given the vast diversity of compounds reaching freshwater ecosystems and adverse effects they might cause. In this study, the environmental fate of 73 CEC, comprising sweeteners, stimulants and several pharmaceutical therapeutic classes, and changes in fluvial biofilm photosynthetic parameters were evaluated in a semi-arid urban river receiving diffuse and point sources of pollution (Suquía river, Argentina). Out of the 37 CEC detected, 30 were quantified in surface water (n.d. - 9826 ng/L), 10 in biofilm (n.d. - 204 ng/gd.w.) and 9 in the clay fraction of sediments (n.d. - 64 ng/gd.w.). CEC distribute differently among the 3 matrices: water phase presents the biggest diversity of compounds (14 CEC families), being analgesic/anti-inflammatories the most abundant family. Antibiotics largely predominated in biofilms (7 CEC families), while the stimulant caffeine and some antibiotics where the most abundant in sediments (6 CEC families). Different CEC accumulated in biofilms and sediments upstream and downstream the city, and big shifts of biofilm community occurred downstream WWTP. The shift of biofilm community upstream (F0 > 0) and downstream the WWTP (F0 = 0) shows a sensitive response of F0 to the impact of WWTP. Biofilm photosynthetic parameters responded in less impacted urban sites (sites 1, 2 and 3), where significant correlations were found between ketoprofen and some antibiotics and biofilm parameters. The diversity and amount of CEC found in the urban section of Suquía river alert to the magnitude of point and non-point sources of pollution.

Biological, morphological, and molecular characterization of the baculovirus PlxyMNPV_LBIV-11, and its virulence towards Plutella xylostella, Trichoplusia ni, and Spodoptera frugiperda larvae.

PlxyMNPV_LBIV-11 is an alphabaculovirus strain, isolated from Plutella xylostella larvae. This work characterized this strain at a biological, morphological, and molecular level to evaluate its similarity with other baculoviruses. Its ultrastructure showed a multiple arrangement of nucleocapsids within enveloped virions, all occluded within large cubical polyhedra. PlxyMNPV_LBIV-11 showed infectivity on the Hi5 and Sf9 cell lines, despite these being from heterologous origin. This in vitro infectivity was observed using either BVs or by transfection with genomic DNA. Restriction fragment patterns of PlxyMNPV_LBIV-11, using the enzymes EcoRI, BamHI and HindIII, showed a high relationship with those patterns shown by AcMNPV, except for one or two differential bands with each enzyme. Sequences of core genes lef-8 and lef-9 and the conserved polh gene showed identities ranging from 98 to 100% when compared with those of AcMNPV. Somewhat lower was the sequence identity of the gp64 gene (94%) as compared with those of AcMNPV and PlxyMNPV_CL3, which might be related to the difference in virulence. Besides, the presence of this gene in PlxyMNPV_LBIV-11 indicates that it belongs to group 1 of alphabaculoviruses. A phylogram was estimated with the core and conserved gene sequences, corroborating its high relationship with AcMNPV and PlxyMNPV_CL3. Bioassays were performed with P. xylostella larvae reared on a meridic diet, whose LC50 values indicated lower virulence than AcMNPV when tested against P. xylostella, Spodoptera frugiperda, and Trichoplusia ni larvae. Its virulence against S. frugiperda was only seven times lower than AcMNPV. Its potential as a biological control agent is discussed.

Molecular and Toxicological Characterization of a Bacillus thuringiensis Strain Expressing a Vip3 Protein Highly Toxic to Spodoptera frugiperda (Lepidoptera: Noctuidae).

The characterization of the Bacillus thuringiensis (Berliner) LBIT-418 strain was based on a previous work which indicated its high insecticidal potential. Therefore, toxicological, molecular, and biochemical characterizations were conducted in this work to identify its unique features and its potential to be developed as a bioinsecticide. This strain, originally isolated from a healthy mosquito larva, was identified within the subspecies kenyae by sequencing of the hag gene and by the multilocus sequence typing (MLST) technique. Genes cry1Ac2, cry1Ea3, cry2Aa1 and cry2Ab4, and a cry1Ia were detected in its genome, in addition to a vip3Aa gene. In this research, the latter protein was successfully cloned, expressed, and purified and showed high toxicity towards the fall armyworm, Spodoptera frugiperda (J.E. Smith), fourth instar larvae in bioassays using the microdroplet ingestion technique, estimating an LD50 of 21.38 ng/larva. Additional bioassays were performed using the diet surface inoculation technique of the strain's spore-crystal complex against diamondback moth larvae, Plutella xylostella (Linnaeus), estimating an LC50 of 10.22 ng/cm2. Its inability to produce ß-exotoxin was demonstrated by bioassays against the nematode Caenorhabditis elegans Maupas and by HPLC analysis. These results support the high potential of this strain to be developed as a bioinsecticide.

Virulence and genetic characterization of six baculovirus strains isolated from different populations of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda (Smith, 1797), is a polyphagous, voracious, and economically important agricultural pest. Biological control of FAW is a strategy that must be further explored. This study evaluated six baculovirus strains isolated from infected FAW larvae from Mexico, Argentina, Honduras, and the United States. Five alphabaculoviruses (SfNPV-An2, SfNPV-Arg, SfNPV-Fx, SfNPV-Ho, and SfNPV-Sin) and one betabaculovirus (SfGV-RV) were tested against FAW larvae, showing a wide diversity of virulence levels among strains when their estimated LC50s were compared, being SfNPV-Arg, SfNPV-Ho and SfNPV-Fx more virulent than SfNPV-An2, SfNPV-Sin, and SfGV-RV. To determine any virulence difference in vitro studies of these isolates, Sf9 cell cultures were used. Interestingly, only ODVs from four of the test SfNPV strains showed infectivity on Sf9 cell cultures, and some differences in virulence were observed. Genomic restriction analyses and partial sequences of lef-8, lef-9, and polh/granulin genes showed little variability among alphabaculoviruses, both, among them and with previously reported sequences. However, sequences from SfGV-RV were closer to previously reported sequences from the SfGV-VG008 strain than the SfGV-Arg and SfGV-VG014 strains. The great difference in the in vivo virulence was not correlated with great similarity among the isolates. The characterization of these six baculovirus isolates offers the basis for exploring their potential as biological control agents against S. frugiperda, as well the initial studies on their specific infection mechanisms, evolution, and ecology.

Analysis of the copper removal kinetics of the Philippine giant bamboo (Dendrocalamus asper) in hydroponics.

Copper is the third most utilized metal and is a versatile resource with multiple beneficial uses, but it may also become toxic to aquatic life in excess amount. Thus, there is a need to develop methods to reduce the copper contamination in the environment, particularly in bodies of water. Phytoremediation using Dendrocalamus asper may offer an environment-benign and potentially effective method for copper removal though its effectiveness may take several years to materialize for this technology to become cost-effective. By growing D. asper in synthesized contaminated water and analyzing the change in the copper content of the substrate via atomic absorption spectrophotometry, the removal was found to be optimal at 20 ppm Cu and pH 5. The rate of removal was found to have an order of 2.71 and a kinetic constant of 0.0013 ppm-1.71 day-1. With this, it may be possible to estimate the treatment length of phytoremediation given an initial level of copper contamination and a target concentration.

Use of RNAi as a preliminary tool for screening putative receptors of nematicidal toxins from Bacillus thuringiensis.

Bacillus thuringiensis is a potential control agent for plant-parasitic nematodes. Nematode intestinal receptors for Cry21-type toxins are poorly known. Therefore, a strategy was tested as a primary screening tool to find possible Cry toxin receptors, using a nematicidal Bt strain and the RNAi technique on Caenorhabditis elegans. Six genes encoding intestinal membrane proteins were selected (abt-4, bre-1, bre-2, bre-3, asps-1, abl-1) as possible targets for Cry proteins. Fractions of each selected gene were amplified by PCR. Amplicons were cloned into the L4440 vector to transform the E. coli HT155 (DE3) strain. Transformed bacteria were used to silence the selected genes using the RNAi feeding method. Nematodes with silenced genes were tested with the Bt strain LBIT-107, which harbors the nematicidal protein Cry21Aa3, among others. Results indicated that nematodes with the silenced abt-4 gene were 69.5% more resistant to the LBIT-107 strain, in general, and 79% to the Cry21Aa3 toxin, specifically.

Expression of ChiA74∆sp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins.

Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.

High Virulence of Mexican Entomopathogenic Fungi Against Fall Armyworm, (Lepidoptera: Noctuidae).

Fourteen fungal entomopathogenic strains were isolated from soil samples and infected field-collected fall armyworm larvae, in Guanajuato, Mexico. Isolates were identified by morphology and internal transcribed spacers sequencing. Isolates Ma22, Ma41, and Mr8 showed 99% identity with reference strains (RS) of Metarhizium anisopliae. Isolates Bb9, Bb19, Bb21, Bb40, Bb27, Bb23, and Bb39 showed identity between 99 and 100% with RS of Beauveria bassiana. Isolates Nr1, Nr2, Nr3, and Nr4 showed identity between 98 and 100% with RS of Nomuraea rileyi. Qualitative selection used one concentration (1 × 108 conidia/ml) on fall armyworm eggs and neonate larvae. Strains Ma22, Ma41, and Mr8 showed 100%, and strains Bb39, Bb23, Bb9, Bb40, Bb19, and Bb21 showed 92, 89.2, 87.6, 82.8, 58, and 38% egg mortality, respectively. Bioassays on neonate larvae showed 100% mortality with strains Ma22, Ma41, Mr8, and Bb9. Strains Bb39, Bb19, Bb27, Bb23, Bb21, and Bb40 showed 74, 60, 54, 53, 28, and 19% mortality, respectively. Bioassay estimated LC50s for strains Ma41 at 7.4 × 104, Mr8 at 8.9 × 104, and Ma22 at 10 × 104 conidia/ml, on fall armyworm eggs. LC50s on neonate larvae were estimated at 2.8 × 105, 16 × 105, 26 × 105, and 36 × 105 conidia/ml for strains Ma41, Bb9, Ma22, and Mr8, respectively. Virulence genes mad1 and mad2 were found in Mr8, Ma22, and Ma41, whereas the gen gmact was found only in the strain Ma22. Genes hyd1 and hyd2 were identified in Bb9, Bb19, Bb21, and Bb27. No correlation was observed between the virulence gene detection and the estimated LC50s. Strain Ma41 showed the highest potential to be developed as a bioinsecticide.

New Hybrids Derived from Podophyllic Aldehyde and Diterpenylhydroquinones with Selectivity toward Osteosarcoma Cells.

A new family of molecular hybrids, between cyclolignans related to podophyllic aldehyde and several diterpenylnaphthohydroquinones (DNHQ), was prepared and its biological activity evaluated in several human solid tumor cell lines, which are representative of the most prevalent solid tumors in the Western world. Both cyclolignan and quinone fragments were linked through aliphatic or aromatic spacers. The new hybrid family was evaluated for its cytotoxicity, and it was found that the hybrids were several times more potent against the osteosarcoma cell line MG-63 than against MCF-7 and HT-29 cell lines. The presence of an aromatic ring in the linker gave the most potent and selective agent, improving the cytotoxicity of the parent compounds. Cell cycle studies demonstrated that this hybrid induces a strong and rapid apoptotic effect and arrests cells at the G2/M phase of the cell cycle, in the same way that the parent compound podophyllic aldehyde does.

Cytotoxic Terphenyl Neolignans from Fungus Terana coerulea: New Natural Corticins D and E. and Revised Structure for Corticin A.

The cobalt crust fungus Terana coerulea (Phanerochaetaceae family) was selected for a bio-guided study after an ethnobotanical survey at the Irati's Forest (Navarra, Spain) for its local use as antibiotic. Six extracts of increasing polarity, from hexane to hot water, were obtained from powdered dry fungi and tested for cytotoxicity against four human tumour cell lines and one non-tumour primary cell culture. From the most cytotoxic, EtOAc extract, we isolated and identified three terphenyl neolignans: two of them new natural products, named corticins D and E, and one previously described as corticin A, whose earlier structure has been revised. Their structural elucidation and biological evaluation as cytotoxic agents are described.

Transformation kinetics of fermented milk using Lactobacillus casei (Lc1) and Streptococcus thermophilus: comparison of results with other Inocula.

Probiotic-based starter cultures are generally used to produce fermented milks with improved characteristics in the final product. In this study, Lactobacillus casei and Streptococcus thermophilus (Lc1-St) were used as the starter inoculum. The transformation kinetics and properties of the final product were compared with systems produced with other inocula. The Lc1-St inoculum delayed the production of lactic acid from 40 to 70 min (depending on temperature and concentration) when compared to Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus (Lb-St) and Lactobacillus johnsonii and Streptococcus thermophilus (La1-St). The Lc1-St inoculum reached the aggregation system faster (30-80 min) than Lb-St (120-210 min) and La1-St (160-220 min), however, the production of exopolysaccharides and organic phosphates was delayed as a consequence of the lack of synergy between Lc1 and St.

Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130 000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of ß-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development.

Novel Poly(3-hydroxybutyrate-g-vinyl alcohol) Polyurethane Scaffold for Tissue Engineering.

The design of new synthetic grafted poly(3-hydroxybutyrate) as composite 3D-scaffolds is a convenient alternative for tissue engineering applications. The chemically modified poly(3-hydroxybutyrate) is receiving increasing attention for use as biomimetic copolymers for cell growth. As of yet, these copolymers cannot be used efficiently because of the lack of good mechanical properties. Here, we address this challenge, preparing a composite-scaffold of grafted poly(3-hydroxybutyrate) polyurethane for the first time. However, it is unclear if the composite structure and morphology can also offer a biological application. We obtained the polyurethane by mixing a polyester hydroxylated resin with polyisocyanate and the modified polyhydroxyalkanoates. The results show that the poly(3-hydroxybutyrate) grafted with poly(vinyl alcohol) can be successfully used as a chain extender to form a chemically-crosslinked thermosetting polymer. Furthermore, we show a proposal for the mechanism of the polyurethane synthesis, the analysis of its morphology and the ability of the scaffolds for growing mammalian cells. We demonstrated that astrocytes isolated from mouse cerebellum, and HEK293 can be cultured in the prepared material, and express efficiently fluorescent proteins by adenoviral transduction. We also tested the metabolism of Ca(2+) to obtain evidence of the biological activity.

Comportamiento del color de discos y restauraciones en resina compuesta sometidos a altas temperatura con fines forenses / Performance of color of discs and restorations in composite resin subject to high temperatures to forensic purposes

El objetivo de este estudio fue determinar el comportamiento del color de discos y restauraciones en resina compuesta sometidos a altas temperaturas con fines forenses. Para ello se realizó un estudio descriptivo de naturaleza pseudoexperimental in vitro que describió los cambios colorimétricos que ocurrieron en 75 discos y 75 dientes restaurados en resina compuesta (Z100 3M-ESPE®), confeccionados en cinco colores (A1, A2, A3, A3.5 y B2), al ser sometidos a la acción de altas temperaturas, con el propósito de establecer parámetros cualitativos a partir del cambio de color de utilidad forense. Los resultados obtenidos permitieron explicar los cambios de color por espectrometría, de tal forma que una resina compuesta, a 200 oC, pierde brillo y matiz; a 400 oC, pierde brillo, matiz y saturación; y a 600 oC, 800 oC y 1.000 oC gana brillo y pierde matiz y saturación. Así, no existieron diferencias significativas en las coordenadas de color L* a* b* al comparar los discos con los dientes restaurados. En conclusión, la interpretación visual del cambio de color y las coordenadas de color L* a* b* a través del uso de un espectrofotómetro puede constituirse en un método comparativo de bajo costo y de aplicación forense al momento de identificar el tipo y el color de un material restaurador en un cadáver o en restos humanos quemados, carbonizados o incinerados, para obtener marcadores positivos durante el cotejo ante mórtem-post mórtem, y estimar la temperatura máxima alcanzada durante la exposición (AU)

The objective for this study was to determine the color behavior of discs and composite resin restorations subjected to high temperature for forensic purposes. For this, a descriptive study of pseudo-experimental nature was made, to describing in vitro colorimetric changes that occur on the surface of 75 disks and 75 class I composite resin restorations (Z100 3M-ESPE®) made in five colors (A1, A2 , A3, A3.5 and B2), when subjected to the action of high temperatures (200 oC, 400 oC, 600 oC, 800 oC y 1,000 oC); in order to set parameters from the color change that can be applied to the forensic dental identification methods in the case of bodies or human remains burnt, charred or incinerated. The color changes can be explained by spectrometry. A composite resin subjected to 200 oC loses brightness and hue; to 400 oC wins brightness, hue and saturation; to 600 oC, 800 oC and 1,000 oC lost brightness, hue and saturation. No significant differences in the color coordinates L * a * b * to compare discs with the restored teeth, so the test bodies were suitable to test with the commercial composite resin system ESPE® 3M-Z100. In conclusion, the visual interpretation of color change and the L * a * b * color coordinates through the use of a spectrometer becomes a comparative forensic method application inexpensive when identifying the type and color a restorative material in a human remains burnt, charred or incinerated, to obtain positive markers for the antemortem-postmortem comparison and estimate the maximum temperature reached (AU)

Effects of different arachidonic acid supplementation on psychomotor development in very preterm infants; a randomized controlled trial.

BACKGROUND & AIMS: Nutritional supplementation with polyunsaturated fatty acids is important in preterm infants neurodevelopment, but it is not known if the omega-6/omega-3 ratio affects this process. This study was designed to determine the effects of a balanced contribution of arachidonic acid in very preterm newborns fed with formula milk. METHODS: This was a randomized trial, in which newborns <1500 g and/or <32 weeks gestational age were assigned to one of two groups, based on the milk formula they would receive during the first year of life. Initially, 60 newborns entered the study, but ultimately, group A was composed of 24 newborns, who were given formula milk with an ω-6/ω-3 ratio of 2/1, and Group B was composed of 21 newborns, given formula milk with an ω-6/ω-3 ratio of 1/1. The infants were followed up for two years: growth, visual-evoked potentials, brainstem auditory-evoked potentials, and plasma fatty acids were periodically measured, and psychomotor development was assessed using the Brunet Lézine scale at 24 months corrected age. A control group, for comparison of Brunet Lézine score, was made up of 25 newborns from the SEN1500 project, who were fed exclusively with breast milk. RESULTS: At 12 months, arachidonic acid values were significantly higher in group A than in group B (6.95 ± 1.55% vs. 4.55 ± 0.78%), as were polyunsaturated fatty acids (41.02 ± 2.09% vs. 38.08 ± 2.32%) achieved a higher average. Group A achieved a higher average Brunet Lézine score at 24 months than group B (99.9 ± 9 vs. 90.8 ± 11, p =0.028). The Brunet Lézine results from group A were compared with the control group results, with very similar scores registered between the two groups (99.9 ± 9 vs. 100.5 ± 7). There were no significant differences in growth or evoked potentials between the two formula groups. CONCLUSIONS: Very preterm infants who received formula with an ω-6/ω-3 ratio of 2/1 had higher blood levels of essential fatty acids during the first year of life, and better psychomotor development, compared with very preterm newborns who consumed formula with an ω-6/ω-3 of 1/1. Therefore, formula milk with an arachidonic acid quantity double that of docosahexaenoic acid should be considered for feeding very preterm infants. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02503020.

Radiation-induced graft polymerization of chitosan onto poly(3-hydroxybutyrate).

Chitosan is among the most studied biopolymers and offers important advantages, such as biodegradability, biocompatibility and nontoxicity. In this study, this polysaccharide was grafted onto poly(3-hydroxybutyrate) using the simultaneous gamma-irradiation-initiated polymerization method. The polyester was immersed in diverse solvents, which allowed the preparation of graft copolymers with different yields and crystallinities. A successful synthesis and the estimation of the degree of crystallinity were verified by spectroscopic and calorimetric techniques. The most suitable method was found to be the thermoanalytical approach because it displayed a linear relationship between the degree of crystallinity and the increasing degree of grafting. The results also indicated that the lowest degree of grafting was seen for acetic acid (14.27%), while the highest degree corresponded to ethyl acetate (32.11%). The mechanism of grafting was proposed on the basis of the experimental results.

Influence of the interaction between phosphate and arsenate on periphyton's growth and its nutrient uptake capacity.

Periphyton communities grown in microcosms were studied under the exposure to different arsenate (As) and phosphate (P) regimes with the aim of revealing the effect of chronic exposure to As on periphyton physiological and structural characteristics. Also, we aimed to study periphyton changes on sensitivity to As, exposed to different P and As regimes. As affected structural and functional parameters of periphyton communities starved of P, inhibiting algal growth, photosynthetic capacity, changing community composition and reducing the ability of the community to retain P. The effects of As on these parameters were only detected in P starved communities, showing that chronic exposure to As led to changes in the photosynthetic apparatus under the conditions of P-limitation, but not when P-availability was higher. This fact reveals a lower toxicity and/or a higher adaptation of the P-amended community. Intracellular As contents were higher in communities starved of P. However, As tolerance was only induced by the combination of As and P but not by As or P alone indicating that tolerance induction may be an ATP-dependent mechanism. This study reveals that chronic exposure of natural communities to environmentally realistic As concentrations will damage periphyton communities affecting key ecosystem processes, as P uptake, leading to changes in stream ecosystems, as these organisms play a key role in nutrient cycling through nutrient uptake and transfer to higher trophic levels.

Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use.

BACKGROUND: The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. RESULTS: Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC50s of 67.30 ng/cm² and 41.45 ng/cm², respectively. CONCLUSIONS: Alkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.