Injectable chitosan-platelet-rich plasma implants to promote tissue regeneration: in vitro properties, in vivo residence, degradation, cell recruitment and vascularization.

The purpose of this study was to develop freeze-dried chitosan formulations that can be solubilized in platelet-rich plasma (PRP) to form injectable implants for tissue repair. A systematic approach to adjust formulation parameters, including chitosan number average molar mass (Mn ), chitosan concentration and lyoprotectant concentration, was undertaken to identify compositions that would rapidly (< 1 min) and completely solubilize in PRP, would have paste-like handling properties upon solubilization and coagulate rapidly (< 5 min) to form solid chitosan-PRP hybrid implants that are stable and homogenous. Freeze-dried cakes containing calcium chloride, as well as distinct chitosan Mn , chitosan concentration and lyoprotectant concentration, were prepared. PRP was used to solubilize the freeze-dried cakes and assess in vitro and in vivo performance, the latter as dorsal subcutaneous injections into New Zealand White rabbits. Freeze-dried polymer formulations containing low and medium chitosan Mn and concentrations were rapidly and completely solubilized in PRP. The paste-like chitosan-PRP mixtures coagulated quickly to form solid chitosan-PRP hybrids, which retracted much less than PRP-only controls. Homogeneous dispersion of chitosan within the hybrid clots was strongly dependent on chitosan Mn , and occurred only with medium Mn chitosan. Chitosan-PRP hybrid clots were resident subcutaneously in vivo until at least 2 weeks while PRP controls were quickly degraded in one day. Compared to PRP alone, chitosan-PRP hybrids had much greater capacity to induce local cell recruitment accompanied by angiogenesis, suggesting a strong potential for their use in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Assessment of in vitro pharmacological effect of Neotropical Piperaceae in GABAergic bioassays in relation to plants traditionally used for folk illness by the Yanesha (Peru).

ETHNOPHARMACOLOGICAL RELEVANCE: A previous pilot ethnobotanical and ethnopharmacological study with the Q'echi׳ Maya identified the family Piperaceae, as an important taxonomic group traditionally used for the treatment of epileptic and culture-bound anxiety disorders and possessing activity in the GABA system. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae were collected including 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an indigenous Amazonian group. MATERIALS AND METHODS: Two high throughput bioassays were used to quantify the in vitro activity of botanical extracts on the GABA system. RESULTS: Plant extracts demonstrated moderate to high affinity to the γ-aminobutyric acid benzodiazepine (GABA-BZD) receptor. In addition, extracts demonstrated low to moderate activity in the inhibition of the GABA-transaminase, with select plants exhibiting significant activity. Plants indicated by the Yanesha showed comparable activity to the other Piperaceae plants collected. Piper cremii was the most active plant in the GABA-BZD receptor assay, and Drymaria cordata (Caryophyllaceae) in the GABA-T assay. CONCLUSION: The study provides evidence that there is a pharmacological basis behind the use of plants in the treatment of susto and mal aire in both Central and South America, and we propose that the possible mechanism of action includes an interaction with the GABA-T enzyme and/or the GABAA-BZD receptor.

Cellulose nanocrystal iridescence: a new model.

A homogeneous aqueous dispersion of cellulose nanocrystals (CNs) that is left to evaporate in a Petri dish self-organizes into smectic liquid crystals that are actually liquid multilamellar structures. As evaporation proceeds, the liquid multilamellar structures solidify to become a solid multilamellar film. Each solid lamella is in the submicrometer range, and its iridescence is easily explained by classical light interference. A careful inspection of each solid lamella revealed long, oriented arrays of colloids. Interestingly, the array orientation is generally the same for each superposed layer. This is exceptional because the stratification appears first in the liquid, and the solid colloids are formed in each stratum at the very end of the process. Our findings are supported by optical, atomic force, and electron microscope observations and by laser diffraction observations. The multilamellar solid film model is easier to engineer than the helical model currently used to explain the iridescence and optical activities of CN solid films. This new understanding should promote the industrial production of colorful CN coatings and inks as a green alternative for decades to come.

Constraining the Sheffield dynamic global vegetation model using stream-flow measurements in the United Kingdom.

The biospheric water and carbon cycles are intimately coupled, so simulating carbon fluxes by vegetation also requires modelling of the water fluxes, with each component influencing the other. Observations of river streamflow integrate information at the catchment scale and are widely available over a long period; they therefore provide an important source of information for validating or calibrating vegetation models. In this paper, we analyse the performance of the Sheffield dynamic global vegetation model (SDGVM) for predicting river streamflow and quantifying how this information helps to constrain carbon flux predictions. The SDGVM is run for 29 large catchments in the United Kingdom. Annual streamflow estimates are compared with long time-series observations. In 23 out of the 29 catchments, the bias between model and observations is less than 50 mm, equivalent to less than 10% of precipitation. In the remaining catchments, larger errors are because of combinations of unpredictable causes, in particular various human activities and measurement issues and, in two cases, unidentified causes. In one of the catchments, we assess to what extent a knowledge of annual streamflow can constrain model parameters and in turn constrain estimates of gross primary production (GPP). For this purpose, we assume the model parameters are uncertain and constrain them by the streamflow observations using the generalized likelihood uncertainty estimation method. Comparing the probability density function of GPP with and without constraint shows that streamflow effectively constrains GPP, mainly by setting a low probability to GPP values below about 1100 g C-1 m2 yr-1 . In other words, streamflow observations allow the rejection of low values of GPP, so that the potential range of possible GPP values is almost halved.

Elastic consequences of a single plastic event: a step towards the microscopic modeling of the flow of yield stress fluids.

With the eventual aim of describing flowing elasto-plastic materials, we focus here on the elementary process of such a flow, a plastic event, and compute the long-range perturbation it elastically induces in a medium submitted to a global shear strain. We characterize the effect of a nearby wall on this perturbation, and quantify the importance of finite-size effects. Although most of our explicit formulae refer to 2D situations, our statements hold for 3D situations as well.

Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms.

A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3. Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin. The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units.

Structural analysis and physical mapping of a pericentromeric region of chromosome 5 of Arabidopsis thaliana.

The Arabidopsis thaliana CIC YAC 2D2, 510 kb long and containing a small block of 180 bp satellite units was subcloned after EcoR1 digestion in the pBluescript plasmid. One of these clones was mapped genetically in the pericentromeric region of chromosome 5. The analysis of 40 subclones of this YAC showed that they all contain repeated sequences with a high proportion of transposable elements. Three new retrotransposons, two Ty-3 Gypsy-like and one Ty-1 Copia, were identified in addition to two new tandem-repeat families. A physical map of the chromosome 5 pericentromeric region was established using CIC YAC clones, spanning around 1000 kb. This contig extends from the CIC YAC 9F5 and 7A2 positioned on the left arm of chromosome 5 to a 5S rDNA genes block localized by in-situ hybridization in the pericentromeric region. Hybridization of the subclones on the CIC YAC library showed that some of them are restricted to the pericentromeric region of chromosome 5 and represent specific markers of this region.

A YAC contig map of Arabidopsis thaliana chromosome 3.

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low-copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large-scale DNA sequencing of chromosome 3.

Capillary electrophoresis determinative and LC-MS confirmatory method for screening selected imidazolinone herbicides from soil.

Residues of imazapyr, imazamox, imazapic, imazethapyr, imazaquin, and imazamethabenz (meta and para) are extracted from soil with 0.5 N sodium hydroxide. The pH is adjusted to 2.0-2.2, and the resulting precipitate is filtered. Compounds are trapped onto a tC18 solid-phase extraction (SPE) cartridge, then eluted from the cartridge and passed through a strong anion exchange (SAX) SPE cartridge onto a benzenesulfonic acid strong cation exchange (SCX) cartridge using ethyl acetate. After eluting the analytes from the SCX cartridge using saturated potassium chloride in methanol, the solution is evaporated and redissolved in 1% formic acid in water. The sample is then desalted using a tC18 SPE cartridge and eluted with methanol. After evaporating the methanol to dryness, the compounds are partitioned from acidic solution (pH 3.5) into methylene chloride. The methylene chloride is evaporated to dryness and the residues are then dissolved in Milli-Q water (Millipore, Bedford, MA, U.S.A.) in preparation for analysis by capillary electrophoresis. Results are calculated by direct comparison of the sample peak heights to the peak heights of bracketing standards. The validated sensitivity of the method (LOQ, limit of quantitation) is 2.0 ppb for each compound. Confirmation for individual residues greater than 2.0 ppb is provided by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) of the final extract.

Evolution of SINE S1 retroposons in Cruciferae plant species.

The S1 element is a plant short interspersed element (SINE) that was first described and studied in Brassica napus. In this work, we investigated the distribution and the molecular phylogeny of the S1 element within the Cruciferae (= Brassicaceae). S1 elements were found to be widely distributed within the Cruciferae, especially in species of the tribe Brassiceae. The molecular phylogeny of S1 elements in eight Cruciferae species (Brassica oleracea, Brassica rapa, Brassica napus, Brassica nigra, Sinapis, arvensis, Sinapis pubescens, Coincya monensis, and Vella spinosa) was inferred using 14-36 elements per species. Significant neighbor-joining and maximum-parsimony phylogenetic clusters, supported by high bootstrap P values and/or represented in 100% of the most-parsimonious trees, were observed for each species. Most of these clusters probably correspond to recent species-specific bursts of S1 amplification. Since these species diverged recently, S1 amplification in Cruciferae plants is proposed to be a highly dynamic process that could contribute to genome rearrangements and eventually lead to reproductive isolation. S1 sequence analysis also revealed putative gene conversion events that occurred between different S1 elements of a given species. These events suggest that gene conversion is a minor but significant component of the molecular drive governing S1 concerted evolution.

Plant S1 SINEs as a model to study retroposition.

The S1 element is a plant SINE (Short INterspersed Element) that was first described and studied in Brassica napus and is widely distributed among Cruciferae, especially in species of the Brassiceae tribe. We propose that S1 amplification in Cruciferae could represent a good eukaryotic model to study retroposition. This is based on the fact that S1 elements share clear structural and evolutionary characteristics with mammalian SINEs but are present in a much lower copy number (500 loci by haploid genome for the S1 element in B. napus compared to 700,000 loci by haploid genome for the Alu element in human). This low copy number allows the characterization of a large portion of SINEs from a given plant species. This can lead to a more precise understanding of the evolutionary history of SINE amplification and can more easily allow an evaluation of the impact of retroposition on the evolution of that species. It can also lead more rapidly to the characterization of genomic elements active in transcription and retroposition so that the cellular control of these elements can be addressed. Finally, we show that the study of S1 insertion sites can reveal information on the RNA reverse transcription and integration step of the retroposition process.

A transcriptional analysis of the S1Bn (Brassica napus) family of SINE retroposons.

S1Bn is a plant short interspersed element (SINE) whose amplification probably involves the reverse transcription of an RNA intermediate. In this report, we identified and characterized S1Bn transcripts from different Brassica napus tissues. Despite the presence of a consensus internal POL III promoter in a large number of genomic S1Bn elements, we observed that S1Bn transcripts are rare in B. napus cells. The use of two very sensitive methods (RT-PCR and RACE PCR) allowed the characterization of 102 independent S1Bn cDNA clones from three different tissues (shoot, root and callus). From this analysis, we conclude that the majority of S1Bn transcripts probably result from a small number of cotranscriptional events where an S1Bn element is transcribed due to its presence in a POL II transcriptional unit. Specific POL III RNA transcripts, initiating at the first 5' nucleotide of the DNA element, are also present in the tested tissues and possibly result from the transcriptional activity of as few as three genomic elements. Two of these transcripts could represent master transcripts responsible for the amplification of S1Bn subfamilies. We also observed that the population of specific POL III transcripts varies among the three tested tissues and that some transcripts appear completely tissue-specific.

DNA regions flanking the major Arabidopsis thaliana satellite are principally enriched in Athila retroelement sequences.

An analysis of Arabidopsis thaliana heterochromatic regions revealed that genomic sequences immediately flanking the major 180 bp satellite are essentially made of middle repetitive sequences and that most of these sequences correspond to defective Athila retroelements. Using YAC and lambda clones, we evaluated the distribution of Athila elements in the Arabidopsis genome and showed that, despite the presence of numerous euchromatic copies, these elements are especially concentrated in or near heterochromatic regions. Sequencing of the various DNA transitions between satellite and Athila repeats provides strong evidence that most of the heterochromatic elements retrotransposed directly into 180 bp satellite clusters.

A promoter identified in the 3' end of the Ac transposon can be activated by cis-acting elements in transgenic Arabidopsis lines.

In experiments directed to develop a promoter trap strategy in Arabidopsis, using a Ds chimaeric element containing a promoterless beta-glucuronidase (GUS) gene, we identified a promoter in the 3' end region of the Ac transposable element. The promoter initiates most of the transcripts at coordinate 4250 in the Ac sequence and is oriented towards the internal part of the element. When fused to a promoterless GUS gene, the promoter allows transient expression in Arabidopsis leaves. After stable integration into the Arabidpsis genome, no GUS activity was observed in most of the transformed lines analysed. Only two of them exhibited different tissue-specific GUS expression. When a CaMV 35S promoter was introduced into the transformation vector, downstream to the reporter gene, a high level of GUS activity was observed in all the transformants. These results strongly suggest that the promoter is not normally expressed at a significant level in Arabidopsis transformed lines except when activated by neighbouring cis-acting enhancer elements. This opens an interesting possibility for using this promoter to develop 'enhancer trap' strategies in Arabidopsis. Since only one Ac transcript, initiating in the 5' end region of the element has been reported to date in maize, the putative biological function of the promoter remains an open question.

Athila, a new retroelement from Arabidopsis thaliana.

An analysis of Arabidopsis thaliana heterochromatic regions allowed the identification of a new family of retroelements called Athila. These 10.5 kb elements, representing ca. 0.3% of the genome, present several features of retrotransposons and retroviruses. Athila elements are flanked by 1.5 kb long terminal repeats (LTR) that are themselves bounded by 5 bp perfect inverted repeats. These LTRs start and end with the retroviral consensus 5'TG...CA3' nucleotides. A putative tRNA-binding site and a polypurine tract are found adjacent to the 5' and 3' LTR respectively. The central domain is composed of two long open reading frames (ORFs) of 935 and 694 amino acids. Despite several indications of recent transposition activity, the translation of these ORFs failed to reveal significant homology with proteins associated to retrotransposition. We suggest that the Athila family could result from the transduction and dispersion of a cellular gene by a retrotransposon.

The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana.

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.

The viability of Scenedesmus bicellularis cells immobilized on alginate screens following nutrient starvation in air at 100% relative humidity.

The viability of algal cells immobilized on screens and starved in a water-saturated air stream was studied at the laboratory scale. This new process for wastewater biotreatment has been developed using immobilized cells, which were starved in air, to obtain a high rate of nutrient removal. A unicellular green microalgae, Scenedesmus bicellularis, was isolated from secondary decantation tanks at an urban wastewater treatment plant and grown in a synthetic medium for 12 days. The cells were then concentrated by centrifugation and immobilized on alginate screens. The screens were then inserted in a photochamber saturated at 100% relative humidity and subjected to a photoperiod of 16 h in the light and 8 h in the dark, with an illumination of 150 muE m(-2) s(-1) provided by fluorescent lamps. After 48 h of nutrient starvation, the immobilized cells were used for the removal of ammonium and orthophosphate from a synthetic secondary wastewater effluent in a plexiglass reactor. During the sequential operation of starvation followed by incubation in the presence of nutrients, fast growth of viable cells in the gel matrix was obtained and there was no appreciable decay of chlorophyll a or cell activity. When these immobilized and starved cells were incubated in wastewater, ammonium (NH(4) (+)) and orthophosphate (PO(4) (3-)) ions were quickly taken up from medium. After three successive 2-h exposures to wastewater, immobilized algal cells were freed by dissolving the Ca-alginate with phosphate as 0.2 M Na(3)PO(4) and resuspended in fresh culture medium. Results indicate that free cells transferred to rich medium remained viable, but the growth rate revealed that the viable cells decreased their culturability. (c) 1995 John Wiley & Sons, Inc.

Determination of hydramethylnon residues in grass by liquid chromatography with confirmation by liquid chromatography/mass spectrometry.

An improved method for determination of hydramethylnon residues in pasture grass is described. The method uses (1) the hydrochloride salt of hydramethylnon to improve its water solubility and (2) an acid-methanol precipitation to remove chlorophylls while leaving the analyte in solution. The liquid chromatographic method has a validated sensitivity of 0.05 ppm with controls showing < 0.004 ppm. The overall average recovery from 0.05 to 0.50 ppm was 98%, with a standard deviation of 11%. Samples showing a positive response (0.05 ppm or higher) and requiring mass spectrometric confirmation are directly amenable to liquid chromatography/mass spectrometry without additional sample preparation.

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci.

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.

An analysis of retroposition in plants based on a family of SINEs from Brassica napus.

The identification of a family of SINE retroposons dispersed in the genome of oilseed rape Brassica napus has provided the basis for an evolutionary analysis of retroposition in plants. The repetitive elements (called S1Bn) are 170 bp long and occupy roughly 500 loci by haploid genome. They present characteristic features of SINE retroposons such as a 3' terminal A-rich region, two conserved polymerase III motifs (box A and B), flanking direct repeats of variable sizes, and a primary and secondary sequence homology to several tRNA species. A consensus sequence was made from the alignment of 34 members of the family. The retroposon population was divided into five subfamilies based on several correlated sets of mutations from the consensus. These precise separations in subfamilies based on "diagnostic" mutations and the random distribution of mutations observed inside each subfamily are consistent with the master sequence model proposed for the dispersion of mammalian retroposons. An independent analysis of each subfamily provides strong evidence for the coexpression of at least three subfamily master sequences (SMS). In contrast to mammalian retroposition, diagnostic positions are not shared between SMS. We therefore propose that SMS were all derived from a general master sequence (GMS) and independently activated for retroposition after a variable period of random drift. Possible models for plant retroposition are discussed.