A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels

Low-molecular-weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non-sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non-sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.     

Mixing Bandt-Pompe and Lempel-Ziv approaches: another way to analyze the complexity of continuous-state sequences

In this paper, we propose to mix the approach underlying Bandt-Pompe permutation entropy with Lempel-Ziv complexity, to design what we call Lempel-Ziv permutation complexity. The principle consists of two steps: (i) transformation of a continuous-state series that is intrinsically multivariate or arises from embedding into a sequence of permutation vectors, where the components are the positions of the components of the initial vector when re-arranged; (ii) performing the Lempel-Ziv complexity for this series of ‘symbols’, as part of a discrete finite-size alphabet. On the one hand, the permutation entropy of Bandt-Pompe aims at the study of the entropy of such a sequence; i.e., the entropy of patterns in a sequence (e.g., local increases or decreases). On the other hand, the Lempel-Ziv complexity of a discrete-state sequence aims at the study of the temporal organization of the symbols (i.e., the rate of compressibility of the sequence). Thus, the Lempel-Ziv permutation complexity aims to take advantage of both of these methods. The potential from such a combined approach – of a permutation procedure and a complexity analysis – is evaluated through the illustration of some simulated data and some real data. In both cases, we compare the individual approaches and the combined approach.     

Preparation of Biodegradable Polylactide Microparticles via a Biocompatible Procedure

PLA MPs are prepared via a novel and toxic‐chemical‐free fabrication route using ethyl lactate, a green solvent and FDA‐approved aroma. MPs are obtained by a solution jet break‐up and solvent displacement method. Adjusting flow parameters allows the tuning of MPs size between 60 and 180 µm, with reduced polydispersity. Morphological analysis shows microporous particles with Janus‐like surface. A fluorophore is successfully loaded into the MPs during their formation step. This versatile green solvent‐based procedure is proven to be suitable for drug encapsulation and delivery applications. The method may be extended to different droplet generation techniques.

     

Passively Q-switched Tm:YLF laser

Stable passive Q-switching of a Tm: LiYF4 laser is obtained using polycrystalline Cr2+: ZnS as a saturable absorber. The achieved maximum pulse energy of 0.9 mJ and peak power of 65 kW for a pulse duration of ～14 ns represent substantial improvement and highest values for a passively Q-switched diode-pumped Tm laser operating at ～1.9 μm.     

Identification,Quantification, and Characterization of the Phenolic Fraction of Brunfelsia grandiflora: In Vitro Antioxidant Capacity

Brunfelsia grandiflora is an ancient plant widely used for its promising medicinal properties, although little explored scientifically. Despite being a rich source of phenolic compounds responsible in part for the proven anti-inflammatory activity, its characterization has not been carried out to date. The present work deals with the exhaustive identification and quantification of its phenolic fraction, along with its antioxidant activity. Decoction resulting from the bark as fine powder was filtered and lyophilized, and polyphenols were extracted from the resulting product by aqueous-organic solvents. Seventy-nine polyphenols were identified using LC-MSⁿ. Hydroxycinnamates was the most abundant group of compounds (up to 66.8%), followed by hydroxycoumarins (15.5%), lignans (6.1%), flavonols (5.7%), phenolic simples (3.1), gallates (2.3%), flavanols (0.3%), and flavanones (0.2%). About 64% of the characterized phenols were in their glycosylated forms. The quantification of these phytochemicals by LC-QToF showed that this medicinal plant contained 2014.71 mg of phenolic compounds in 100 g dry matter, which evidences a great antioxidant potency determined by ABTS and DPPH assays. Therefore, Brunfelsia grandiflora represents an important source of polyphenols which supports its therapeutic properties scientifically proven.     

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species

Polymerase chain reation (PCR) fingerprints are used to characterize and recognize bacteria and are generally obtained using universal primers that generate an array of DNA amplicons, which can be separated by electrophoresis. Universal primers 8F and 1491 R have been used to amplify specifically 16S rDNA. We have used these primers at an annealing temperature of 50 degrees C. Agarose gel electrophoresis of PCR products revealed several bands. The band pattern of each bacterial species was different and the strains belonging to the same species shared an identical pattern. The patterns obtained did not show variations with plasmid DNA content or the growth stage of the bacteria. The peculiarity of the randomly amplified polymorphic DNA (RAPD) described in this work lies in the use of two large primers (proximately 20 nt) to obtain the pattern, since normally a only smaller primer is used, and in the new application for the primers used to amplify 16S rDNA. This new procedure, called two primers (TP)-RAPD fingerprinting, is thus rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species.     

Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples

The random intergenic spacer analysis is a recently developed technique for the study of microbial populations. The bacterial intergenic spacer (ITS) is located between 16S rRNA and 23S rRNA genes and presents different length and sequence among bacterial species. Therefore, the amplicons can be separated by electrophoresis commonly performed at low voltage during several hours. Although this technique is especially useful for unculturable microorganisms, it has not been applied before to clinical sample analysis. As these samples have a limited number of bacterial species, the size of the gels may be reduced to facilitate their handling and to reduce the running time. To obtain maximum separation among the ITS bands, we analysed in this work different electrophoretical conditions including staircase electrophoresis, a technique based on the application of several voltage steps. The results obtained showed a different behaviour of the electrical resistance during the performance of submarine horizontal and vertical staircase electrophoresis. In the first case the resistance decreased during most of the running time whereas in the second case it increased. Here, we show that the performance of horizontal staircase electrophoresis reduces the running time more than 80% with respect to conventional electrophoresis at low voltages. This procedure was applied to the separation of ITS bands from bacterial DNA present in a tissue from a vocal cord biopsy. The sequencing of these bands allowed their identification. This new procedure may be very useful in the rapid diagnosis of bacteria present in human, animal and plant tissues.     

Solid-phase synthesis of polysubstituted piperidines by imino-Diels-Alder cycloaddition of 2-amino-1,3-butadienes with solid-supported imines

[reaction: see text] The solid-phase imino-Diels-Alder reaction of 2-amino-1,3-butadienes with solid-supported imines is described. The reaction furnishes 4-piperidones and 4-aminopiperidines with high diastereoselectivity and with very good yields and purity after the release from the solid support. The possibility of introducing variations in both cycloaddition partners gives rise to substituted piperidines with up to five elements of diversity.