Bioengineering bacterial outer membrane vesicles as delivery system for RNA therapeutics targeted to lung epithelial cytosols

Intact epithelia lining the airways and alveoli in the lung are essential to maintain lung function. Structural or functional damage of epithelial cells leads in severe diseases, including COPD/emphysema, ibrosis or ALI/ARDS. This central role of epithelia in pulmonary diseases identifies these cells as primary candidates for targeted therapy. With the exception of surface-expressed molecules, however, targeting intracellular components is severely restricted due to poor delivery. We aim to overcome this obstacle using topically administered, bioengineered, biocompatible bacterial outer membrane vesicles (OMVs) as recombinant drug delivery systems for novel biopharmaceuticals. Engineering recombinant surface expression of eukaryotic receptor ligands in ClearColi®, a commercial E.coli BL21 (DE3) strain deficient in lipopolysaccharide production, we have used red fluorescent protein reporters to track OMV loading, transgene expression, and eukaryotic cell trafficking. We demonstrate statistically significant differences in the levels of over 700 proteins between differentially engineered and purified OMV preps with additional differences in transcriptome and lipidome consistency. We also characterised visual and particle size differences observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Here we report early bioadhesion and culture of re-differentiated lung epithelia. This project aims to bridge the biotechnological gap in the intracellular biopharmaceutics drug delivery challenge for respiratory epithelia through highly controlled, and scalable bio-nanotechnology process. If successful, our work will unlock intracellular imaging and therapeutics research for respiratory diseases with a significant epithelial component, paving the way for other targeting ligands and potentially non-respiratory indications. cellular uptake results in A549 culture as well as air-liquid interface

Andamento delle prostaglandine F2\u3b1 ematiche nella cavalla in relazione all'esito dell'inseminazione artificiale.Haematic prostaglandin F-2 alpha (PGF(2 alpha)) pattern following successful or -unsuccessful artificial insemination (AI) in the mare

The release of PGF(2 alpha) was monitored during specific phases of the reproductive cycle of artificially inseminated mares. Daily progesterone concentration was measured to detect a possible corpus luteus failure. Five fertile and regularly cycling mares were inseminated with fresh semen from a fertile stallion. the PGF(2 alpha) main metabolite (PGFM) concentration was measured during time windows of 90 minutes at oestrous, 12 hours after the last A.I., and on days 5, 4, 7, 12 following ovulation. three mares were diagnosed pregnant after the first A.I. session and one mare after the second. The analysis of PGFM concentration (both mean values and area under the curve) did not reveal any significant difference among mares or between pregnant and non-pregnant animals. Progesterone concentration was not correlated to PGFM pattern. There was a high variability among animals, both in basal PGFM values (i.e. before A.I.) and in the subsequent determinations. We found no evidence that barren mares have PGFM concentrations different from pregnant ones in critical phases of the oestrous cycle

Using extinctions in species distribution models to evaluate and predict threats: a contribution to plant conservation planning on the island of Sardinia

Recent extinction rates suggest that humans are now causing the sixth mass extinction, and the Mediterranean islands are at the forefront of many of the environmental issues involved. This study provides an alternative approach for investigating documented local plant extinctions that occurred in Sardinia (western Mediterranean) during the last half century. A total of 190 local extinctions of 62 plant species were used to investigate the independent effects of eight ecological and anthropogenic variables and to model the areas of potential extinctions where plant conservation efforts could be focused. If all analysed plant species were considered together, ecological factors explained local extinctions more than anthropogenic factors. The independent effects of each factor considerably varied among species of different lifeforms and altitude ranges. Accordingly, distribution models of local extinctions outscored areas that are potentially rich in plant species with conservation interest, but which are particularly affected by humans. This paper suggests a reproducible, operational framework for analysing which extinction factors may play important roles in similar contexts and where they might be relevant

The genetic structure and diversity of Gentiana lutea subsp. lutea (Gentianaceae) in Sardinia: further insights for its conservation planning

Knowledge of the levels of genetic diversity and of the spatial genetic structure of plant species is important to ensure their effective management and conservation, especially in the case of endangered species. Gentiana lutea L. subsp. lutea is a long-lived plant which occurs in central and southern European mountains. It has a long-standing history of human exploitation, mainly in the liqueur and in the pharmaceutical industries and it is currently listed in the EU Habitats Directive 92/43/EEC Annex V. Mainly due to a prolonged root harvesting, its current distribution range in Sardinia consists of only a few groups of individuals limited to small areas of the Gennargentu massif (Central-Eastern area of the island). In this study, we investigated the levels of genetic diversity and the genetic structure of the species in Sardinia. We used AFLP (amplified fragment length polymorphism) markers to investigate the genetic variability of 182 samples from 13 subpopulations. A total of 433 fragments were detected, of which 75.5% were polymorphic. The levels of genetic diversity were generally high, but they tended to decrease in smaller subpopulations. Of the genetic variability 88% was found within subpopulations, while the genetic structure among them was fairly weak. In order to ensure the survival of these subpopulations, especially the smaller ones, ex situ and in situ management actions should be planned, such as the long term conservation of its seeds in germplasm repositories and their population reinforcements and monitoring

A comprehensive phenotypic investigation of the “Pod-shattering syndrome” in common bean

Seed shattering in crops is a key domestication trait due to its relevance for seed dispersal, yield, and fundamental questions in evolution (e.g., convergent evolution). Here, we focused on pod shattering in common bean (Phaseolus vulgaris L.), the most important legume crop for human consuption in the world. With this main aim, we developed a methodological pipeline that comprises a thorough characterization under field conditions, including also the chemical composition and histological analysis of the pod valves. The pipeline was developed based on the assumption that the shattering trait itself can be treated in principle as a “syndrome” (i.e., a set of correlated different traits) at the pod level. We characterized a population of 267 introgression lines that were developed ad-hoc to study shattering in common bean. Three main objectives were sought: (1) to dissect the shattering trait into its “components,” of level (percentage of shattering pods per plant) and mode (percentage of pods with twisting or non-twisting valves); (2) to test whether shattering is associated to the chemical composition and/or the histological characteristics of the pod valves; and (3) to test the associations between shattering and other plant traits. We can conclude the following: Very high shattering levels can be achieved in different modes; shattering resistance is mainly a qualitative trait; and high shattering levels is correlated with high carbon and lignin contents of the pod valves and with specific histological charaterstics of the ventral sheath and the inner fibrous layer of the pod wall. Our data also suggest that shattering comes with a “cost,” as it is associated with lowpod size, low seed weight per pod, high pod weight, and low seed to pod-valves ratio; indeed, it can be more exaustively described as a syndrome at the pod level. Our work suggests that the valve chemical composition (i.e., carbon and lignin content) can be used for a high troughput phenotyping procedures for shattering phenotyping. Finally, we believe that the application of our pipeline will greatly facilitate comparative studies among legume crops, and gene tagging

Moderate Protein Restriction in Advanced CKD: A Feasible Option in An Elderly, High-Comorbidity Population. A Stepwise Multiple-Choice System Approach

Background: Protein restriction may retard the need for renal replacement therapy; compliance is considered a barrier, especially in elderly patients. Methods: A feasibility study was conducted in a newly organized unit for advanced kidney disease; three diet options were offered: normalization of protein intake (0.8 g/kg/day of protein); moderate protein restriction (0.6 g/kg/day of protein) with a “traditional” mixed protein diet or with a “plant-based” diet supplemented with ketoacids. Patients with protein energy wasting (PEW), short life expectancy or who refused were excluded. Compliance was estimated by Maroni-Mitch formula and food diary. Results: In November 2017–July 2018, 131 patients started the program: median age 74 years (min–max 24-101), Charlson Index (CCI): 8 (min-max: 2–14); eGFR 24 mL/min (4–68); 50.4% were diabetic, BMI was ≥ 30 kg/m2 in 40.4%. Normalization was the first step in 75 patients (57%, age 78 (24–101), CCI 8 (2–12), eGFR 24 mL/min (8–68)); moderately protein-restricted traditional diets were chosen by 24 (18%, age 74 (44–91), CCI 8 (4–14), eGFR 22 mL/min (5–40)), plant-based diets by 22 (17%, age 70 (34–89), CCI 6.5 (2–12), eGFR 15 mL/min (5–46)) (p < 0.001). Protein restriction was not undertaken in 10 patients with short life expectancy. In patients with ≥ 3 months of follow-up, median reduction of protein intake was from 1.2 to 0.8 g/kg/day (p < 0.001); nutritional parameters remained stable; albumin increased from 3.5 to 3.6 g/dL (p = 0.037); good compliance was found in 74%, regardless of diets. Over 1067 patient-months of follow-up, 9 patients died (CCI 10 (6–12)), 7 started dialysis (5 incremental). Conclusion: Protein restriction is feasible by an individualized, stepwise approach in an overall elderly, high-comorbidity population with a baseline high-protein diet and is compatible with stable nutritional status

The Aichi biodiversity target 12 at regional level. An achievable goal?

The Aichi Biodiversity Target 12 aims to prevent the extinction risk of known threatened species and to improve their conservation status by 2020. We present the integrated strategy implemented in the last 10 years for the keystone plant species of Sardinia (Italy, W. Mediterranean Basin), which includes the following activities: conservation status assessment (following the IUCN protocol), ex situ conservation, in situ monitoring and active protection measures. To date, an average of 51.8% of keystone plant species have been subjected to the latter first three activities while, due to the higher costs, only few active conservation measures have been carried out. Considering the activities realised since 2004, we have also predicted the conservation effectiveness towards 2020 and have elaborated an index to evaluate it. Halfway through the strategic plan, we argue that more efforts are needed to guarantee the effective conservation of all threatened plants in Sardinia

Genomic dissection of pod shattering in common bean: mutations at non-orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species

The complete or partial loss of shattering ability occurred independently during the domestication of several crops. Therefore, the study of this trait can provide an understanding of the link between phenotypic and molecular convergent evolution. The genetic dissection of 'pod shattering' in Phaseolus vulgaris is achieved here using a population of introgression lines and next-generation sequencing techniques. The 'occurrence' of the indehiscent phenotype (indehiscent versus dehiscent) depends on a major locus on chromosome 5. Furthermore, at least two additional genes are associated with the 'level' of shattering (number of shattering pods per plant: low versus high) and the 'mode' of shattering (non-twisting versus twisting pods), with all of these loci contributing to the phenotype by epistatic interactions. Comparative mapping indicates that the major gene identified on common bean chromosome 5 corresponds to one of the four quantitative trait loci for pod shattering in Vigna unguiculata. None of the loci identified comprised genes that are homologs of the known shattering genes in Glycine max. Therefore, although convergent domestication can be determined by mutations at orthologous loci, this was only partially true for P. vulgaris and V. unguiculata, which are two phylogenetically closely related crop species, and this was not the case for the more distant P. vulgaris and G. max. Conversely, comparative mapping suggests that the convergent evolution of the indehiscent phenotype arose through mutations in different genes from the same underlying gene networks that are involved in secondary cell-wall biosynthesis and lignin deposition patterning at the pod level

<i>In situ</i> forming biodegradable poly(ε-caprolactone) microsphere systems: a challenge for transarterial embolization therapy. <i>In vitro</i> and preliminary <i>ex vivo</i> studies

<p><b>Background</b>: <i>In situ</i> forming biodegradable poly(ε-caprolactone) (PCL) microspheres (PCL-ISM) system was developed as a novel embolic agent for transarterial embolization (TAE) therapy of hepatocellular carcinoma (HCC). Ibuprofen sodium (Ibu-Na) was loaded on this platform to evaluate its potential for the treatment of post embolization syndrome.</p> <p><b>Methods</b>: The influence of formulation parameters on the size/shape, encapsulation efficiency and drug release was investigated using mixture experimental design. Regression models were derived and used to optimize the formulation for particle size, encapsulation efficiency and drug release profile for TAE therapy. An <i>ex vivo</i> model using isolated rat livers was established to assess the <i>in situ</i> formation of microspheres.</p> <p><b>Results</b>: All PCL-ISM components affected the studied properties and fitting indices of the regression models were high (Radj² = 0.810 for size, 0.964 encapsulation efficiency, and 0.993 or 0.971 for drug release at 30 min or 48 h). The optimized composition was: PCL = 4%, NMP = 43.1%, oil = 48.9%, surfactant = 2% and drug = 2%. <i>Ex vivo</i> studies revealed that PCL-ISM was able to form microspheres in the hepatic arterial bed.</p> <p><b>Conclusions</b>: PCL-ISM system provides a novel tool for the treatment of HCC and post-embolization syndrome. It is capable of forming microspheres with desirable size and Ibu-Na release profile after injection into blood vessels.</p