MicroRNAs delivery into human cells grown on 3D-printed PLA scaffolds coated with a novel fluorescent PAMAM dendrimer for biomedical applications

Many advanced synthetic, natural, degradable or non-degradable materials have been employed to create scaffolds for cell culture for biomedical or tissue engineering applications. One of the most versatile material is poly-lactide (PLA), commonly used as 3D printing filament. Manufacturing of multifunctional scaffolds with improved cell growth proliferation and able to deliver oligonucleotides represents an innovative strategy for controlled and localized gene modulation that hold great promise and could increase the number of applications in biomedicine. Here we report for the first time the synthesis of a novel Rhodamine derivative of a poly-amidoamine dendrimer (G = 5) able to transfect cells and to be monitored by confocal microscopy that we also employed to coat a 3D-printed PLA scaffold. The coating do not modify the oligonucleotide binding ability, toxicity or transfection properties of the scaffold that is able to increase cell proliferation and deliver miRNA mimics (i.e., pre-mir-503) into human cells. Although further experiments are required to optimize the dendrimer/miRNA ratio and improve transfection efficiency, we demonstrated the effectiveness of this promising and innovative 3D-printed transfection system to transfer miRNAs into human cells for future biomedical applications. © 2018, The Author(s)

Biomass furnace for externally fired gas turbine: Development and validation of the numerical model

Externally-fired gas turbines (EFGT) are currently being investigated for co-generation from biomass, because of their ability to deal with low-grade fuels without the complexity of gasification. Main drawbacks of the technology are related to the high thermal stresses experienced by the heat exchanger. The present work proposes a computational fluid dynamics (CFD) analysis of a grate-fired furnace installed in a EFGT cycle, with the purpose to provide a tool for detecting the most critical regions in the furnace. The model is complemented with a process simulation of the entire EFGT cycle. Different approaches for treating the fuel bed and their impact on the CFD analysis are discussed and validated through the availability of in-flame measurements of temperature and chemical species. Predictions indicate the need for a detailed fluid dynamic characterization of the grate region, which was found to largely impact the furnace flow and thermo-chemical fields

The simultaneous insertion of two ligands in gD for the cultivation of oncolytic HSVs in non-cancer cells and the retargeting to cancer receptors

Insertion of a single chain antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Clinical grade virus production can not rely on cancer cells. Recently, we developed a double retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the non-cancer producer Vero-GCN4R cell line, and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more advantageous detargeting strategies were devised, so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of aa 35-39, 214-223, or 219-223, and replacement of the deleted sequences with one of the two ligands. The latter two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best performing recombinants carried a gD deletion as small as 5 amino acids, and grew to titers similar to those exhibited by the singly retargeted R-LM113, and by the non-retargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effectivein vitroandin vivo.IMPORTANCEThere is increasing interest in oncolytic viruses, following FDA and EMA approval of the oncolytic HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in non-cancer cells. Here, we describe a double retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative, one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally-disadvantageous detargeting modifications. The current and accompanying studies teach how to best achieve the clinical-grade cultivation of retargeted oncolytics

Evolution of the methodological quality of controlled clinical trials for myofascial trigger point treatments for the period 1978–2015: A systematic review

Abstract Background The methodological quality of controlled clinical trials (CCTs) of physiotherapeutic treatment modalities for myofascial trigger points (MTrP) has not been investigated yet. Objectives To detect the methodological quality of CCTs for physiotherapy treatments of MTrPs and demonstrating the possible increase over time. Design Systematic review. Methods A systematic search was conducted in two databases, Physiotherapy Evidence Database (PEDro) and Medicine Medical Literature Analysis and Retrieval System online (MEDLINE), using the same keywords and selection procedure corresponding to pre-defined inclusion criteria. The methodological quality, assessed by the 11-item PEDro scale, served as outcome measure. The CCTs had to compare at least two interventions, where one intervention had to lay within the scope of physiotherapy. Participants had to be diagnosed with myofascial pain syndrome or trigger points (active or latent). Results A total of n = 230 studies was analysed. The cervico-thoracic region was the most frequently treated body part (n = 143). Electrophysical agent applications was the most frequent intervention. The average methodological quality reached 5.5 on the PEDro scale. A total of n = 6 studies scored the value of 9. The average PEDro score increased by 0.7 points per decade between 1978 and 2015. Conclusions The average PEDro score of CCTs for MTrP treatments does not reach the cut-off of 6 proposed for moderate to high methodological quality. Nevertheless, a promising trend towards an increase of the average methodological quality of CCTs for MTrPs was recorded. More high-quality CCT studies with thorough research procedures are recommended to enhance methodological quality

Cultural Heritage Leading Urban Futures: Actions and Innovations from ROCK Project

The ROCK project sees historic city centres as laboratories to demonstrate how Cultural Heritage can be an engine of regeneration, sustainable development and economic growth. ROCK approach foresees the systemic and flexible application of a series of role-model practices in the testing sites of three Replicator cities, to turn historic city centres afflicted by physical decay, social conflicts and poor life quality into Creative and Sustainable Districts. This book provides an overview of the project, extracting themes, material and final remarks from the Open Knowledge Week “Cultural Heritage Leading Urban Futuresâ€, held on 27-30 October 2020. Over the past three years, ten ROCK cities – Athens, Bologna, Cluj-Napoca, Eindhoven, Lisbon, Liverpool, Lyon, Skopje, Turin, and Vilnius â€“ together with service providers and knowledge brokers have tested and advanced numerous soft and hard tools, collaborative approaches aimed at shaping sustainable, heritage-led urban futures. This book shows their shared results, best practices and lessons learnt from interdisciplinary research, innovative action, dissemination of knowledge and creation of new synergies at European level

Negative Feedback Regulation of Auxin Signaling by ATHB8/ACL5–BUD2 Transcription Module

ABSTRACT The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and BUSHY AND DWARF2 (BUD2) is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis . Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper ( HD–ZIP ) III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD–ZIP III protein ARABIDOPSIS THALIANA HOMEOBOX8 tightly controlling the expression of ACL5 and BUD2 . In addition, we show that the HD–ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as LIKE AUXIN RESISTANT2 ( LAX2 ) and LAX3 . We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD–ZIP III genes

Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique

Livelli di inquinanti organici persistenti in stenelle (<i>Stenella coeruleoalba</i>) spiaggiate nel nord Sardegna = Concentration of persistent organic pollutants in striped dolphin (<i>Stenella coeruleoalba</i>) stranded in North Sardinia

Long-lived apex predators, such as marine mammals, are particularly at risk from effects of persistent organic pollutants (POPs), e.g. polychlorinated biphenyls (PCBs) and dichlorodiphenylethanes (e.g. DDT), due to bioaccumulation and biomagnification. POPs are lipophilic compounds that tend to accumulate in the lipid-rich blubber. In marine mammals, POPs enter the body almost exclusively through the diet. In the present study, the levels of PCBs and DDTs in the blubber of striped dolphin stranded in North Sardinia were determined. Our results showed that pollutant concentration are related to age and sex of the individuals. Considering the well known harmful consequences of bioaccumulation of POPs in marine mammals, further studies are needed to determine which POPs might be linked to effects on health