Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes.     

D-alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100-23 results in impaired colonization of the mouse gastrointestinal tract

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.     

Quantitative determination by real-time PCR of four vaginalLactobacillus species,Gardnerella vaginalisandAtopobium vaginaeindicates an inverse relationship betweenL. gasseriandL. iners

Background  

Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats forLactobacillus crispatus,L. gasseriandGardnerella vaginalisand developed new formats forAtopobium vaginae,L. inersandL. jenseniito obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years.     

Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy

In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.     

Rice (<i>Oryza sativa</i> L.) Breeding among Hassawi Landrace and Egyptian Genotypes for Stem Borer (<i>Chilo agamemnon</i> Bles.) Resistance and Related Quantitative Traits

Rice stem borer (Chilo agamemnon Bles.) is a primary insect pest of rice and is a major limiting factor to rice production. Breeding for insect-resistant crop varieties has been an economic way of integrated pest management (IPM) as it offers a viable and ecologically acceptable approach. This study was aimed to evaluate rice genotypes for their resistance against rice stem borer. Seven parental genotypes with twenty one F1 crosses were evaluated for genotypic variation in field experiments. Analysis of variance revealed significant differences for the studied traits in almost all crosses and parents. In addition, the mean squares of parents versus their crosses were signifi- cant for stem borer resistance and other associated traits. Moreover, both general combining ability (GCA) and specific combining ability (SCA) variances were highly significant for all characters studied in the F1 generation. Based on GCA, 4 genotypes (Sakha101, Gz6903-3-4-2-1, Gz9577-4-1-1 and Hassawi) exhibited highly significant negative values for stem borer resistance (–0.53, –1.06, –0.18 and –0.49, respectively) indicating they are the best combiners for stem borer resistance. Based on SCA analysis, nine cross combinations showed highly significant negative effects for stem borer resistance. Similarly, the cross Giza178 Hassawi was the best combination with significantly highest value for early maturity. In addition, seven crosses showed highly significant negative SCA for plant height trait. On the other hand, for panicle length, number of primary branches/panicle, panicle weight and 1000-grain weight, seven, four, eight and six crosses showed highly significant positive SCA, respectively. The result further revealed that the non-additive dominance genetic variance was higher than the additive variance for all evaluated traits indicating that non-additive genetic variances have a role in their inheritance. The broad-sense heritability estimates were high for all the studied traits. The stem borer resistance was significantly correlated with panicle weight and 1000-grain weight, which also showed a highly significant correlation with grain yield/plant. Thus these traits can be effectively employed in a breeding program to confer resistance against stem borer infestation in rice. It was further supported by biplot analysis, which clustered these potentially important traits into two quadrants showing their importance in any future breeding program to control stem borer infestation. This study has contributed valuable information for evaluation of genetic diversity in the local rice germplasm and its utilization in futuristic rice genetic improvement programs.     

First volumetric records of airborne Cladosporium and Alternaria spores in the atmosphere of Al Khor (northern Qatar): a preliminary survey

Aerobiologia - Daily monitoring of airborne fungal spores was carried out for the first time in Al Khor city, Qatar, using a Hirst type 7-day recording volumetric spore trap, from May 2017 to May...     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.