Bio-molecular gradient surfaces for biological recognition

The use of protein microfluidic systems is of growing interest for a variety of applications, including but not limited to tissue engineering, drug delivery and biosensors. The means by which to control chemistries on substrates for biological and medical applications is therefore in high demand. Here the creation of a bio-functional gradient on silica and polymeric surfaces using a micro fluidic technique, for the guidance of cell adhesion and functionality, using AFM tools for protein imaging and force spectroscopy investigation is reported. Atomic force microscopy (AFM) is a high resolution microscopic technique highly used in biological investigations, allowing conformational elucidation of protein deposition on the substrate. In this work application of the techniques of AFM, fluorescence microscopy and cell adhesion studies were used to assess the protein deposition along the microfluidic system. From the fluorescence analysis, it was immediately observed that successful protein immobilization on both substrates was achieved. Differences in fluorescence intensity were also registered along the microfluidic channel (start and end point) suggesting a variation in protein adsorption along the channel. The AFM imaging analysis conducted on the same samples revealed a difference in surface coverage considering the injection and end point (from 70% to 14% respectively) of the protein pattern. The difference in protein density registered along the fibronectin pattern was tested using a functionalised probe AFM technique, allowing molecular resolution of ligands in a physiological environment. A difference in the percentage of observed adhesion events was registered considering the start and end point of the microfluidic pattern, from 90% to 37% respectively. This is likely due to the fact that at the higher surface concentration there is a higher probability of the functionalised tip interacting with multiple fibronectin molecules, as confirmed from the presence of multiple adhesions at start point with a higher adhesion force of 82 pN ± 7.4 pN. To complement the AFM force measurements, protein functionality was tested by investigating the cell adhesion, shape and migration on the protein pattern. The fibronectin protein gradient was shown to control cell adhesion and migration along the patterns, demonstrating that this system can be used for biological applications to monitor the cell behaviour using difference protein concentration and cell density all in the same microfluidic channel. The ability to control cell adhesion and migration on substrates could be of significant interest when researching possible applications in future tissue engineering and biological studies. The combination of AFM and fluorescence microscopy techniques for protein density investigation used in this work demonstrated that protein deposition and arrangement on substrate play an important role in cell adhesion and migration studies

Bio-molecular gradient surfaces for biological recognition

The use of protein microfluidic systems is of growing interest for a variety of applications, including but not limited to tissue engineering, drug delivery and biosensors. The means by which to control chemistries on substrates for biological and medical applications is therefore in high demand. Here the creation of a bio-functional gradient on silica and polymeric surfaces using a micro fluidic technique, for the guidance of cell adhesion and functionality, using AFM tools for protein imaging and force spectroscopy investigation is reported. Atomic force microscopy (AFM) is a high resolution microscopic technique highly used in biological investigations, allowing conformational elucidation of protein deposition on the substrate. In this work application of the techniques of AFM, fluorescence microscopy and cell adhesion studies were used to assess the protein deposition along the microfluidic system. From the fluorescence analysis, it was immediately observed that successful protein immobilization on both substrates was achieved. Differences in fluorescence intensity were also registered along the microfluidic channel (start and end point) suggesting a variation in protein adsorption along the channel. The AFM imaging analysis conducted on the same samples revealed a difference in surface coverage considering the injection and end point (from 70% to 14% respectively) of the protein pattern. The difference in protein density registered along the fibronectin pattern was tested using a functionalised probe AFM technique, allowing molecular resolution of ligands in a physiological environment. A difference in the percentage of observed adhesion events was registered considering the start and end point of the microfluidic pattern, from 90% to 37% respectively. This is likely due to the fact that at the higher surface concentration there is a higher probability of the functionalised tip interacting with multiple fibronectin molecules, as confirmed from the presence of multiple adhesions at start point with a higher adhesion force of 82 pN ± 7.4 pN. To complement the AFM force measurements, protein functionality was tested by investigating the cell adhesion, shape and migration on the protein pattern. The fibronectin protein gradient was shown to control cell adhesion and migration along the patterns, demonstrating that this system can be used for biological applications to monitor the cell behaviour using difference protein concentration and cell density all in the same microfluidic channel. The ability to control cell adhesion and migration on substrates could be of significant interest when researching possible applications in future tissue engineering and biological studies. The combination of AFM and fluorescence microscopy techniques for protein density investigation used in this work demonstrated that protein deposition and arrangement on substrate play an important role in cell adhesion and migration studies

antiproliferative activities of artemisia herba alba ethanolic extract in human colon cancer cell line hct116

Artemisia herba-alba (AHE) is a plant commonly used in traditional medicine for the treatment of various ailments. Here, we investigated the antioxidant and antitumor activity of the aqueous and ethanol extracts of AHE in human colon cancer HCT116 cells. The antioxidant activity was measured by DCFH assay, while antitumor effects were assessed by cell viability assays, cell cycle progression by flow cytometry, and DNA fragmentation analysis in addition to investigating the expression of key cell cycle and apoptotic proteins. While the aqueous extract had no antineoplastic effects, the ethanol extract significantly decreased HCT116 viability (IC50 of 51mg/mL at 24 h) and inhibited the production of reactive oxygen species (ROS). Treatment of HCT116 cells with the ethanol extract also caused dramatic increase in the PreG1 population with concomitant decrease in cycling cells, provoked DNA fragmentation, significant increase in the expression levels of p53 and Bax proteins and activated pro-apoptotic caspase-3. The results obtained suggest that the ethanol extract of AHE could be used as an easily accessible source of natural antioxidants and as potential phytochemicals against colon cancer.</p

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Electrospray deposition in vacuum as method to create functionally active protein immobilization on polymeric substrates

We demonstrate in this work the deposition of a large biological molecule (fibronectin) on polymeric substrates in a high vacuum environment using an electrospray deposition system. Fibronectin was deposited and its distribution and structure investigated and retention of function (ability to promote cell adhesion) on return to liquid environment is shown. AFM was used to monitor changes in the morphology of the surface before and after fibronectin deposition, whilst the biological activity of the deposited protein is assessed through a quantitative analysis of the biomolecular adhesion and migration of fibroblast cells to the modified surfaces. For the first time we have demonstrated that using high vacuum electrospray deposition it is possible to deposit large protein molecules on polymeric surfaces whilst maintaining the protein activity. The deposition of biological molecules such as proteins with the retention of their activity onto clean well-controlled surfaces under vacuum condition, offers the possibility for future studies utilizing high resolution vacuum based techniques at the atomic and molecular scale providing a greater understanding of protein–surface interface behaviour of relevance to a wide range of applications such as in sensors, diagnostics and tissue engineering

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

IntroductionIn the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification.MethodsTo do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics.ResultsThese analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes.DiscussionThe presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Antiproliferative activities of Artemisia herba-alba ethanolic extract in human colon cancer cell line (HCT116)

Artemisia herba-alba (AHE) is a plant commonly used in traditional medicine for the treatment of various ailments. Here, we investigated the antioxidant and antitumor activity of the aqueous and ethanol extracts of AHE in human colon cancer HCT116 cells. The antioxidant activity was measured by DCFH assay, while antitumor effects were assessed by cell viability assays, cell cycle progression by flow cytometry, and DNA fragmentation analysis in addition to investigating the expression of key cell cycle and apoptotic proteins. While the aqueous extract had no antineoplastic effects, the ethanol extract significantly decreased HCT116 viability (IC50 of 51mg/mL at 24 h) and inhibited the production of reactive oxygen species (ROS). Treatment of HCT116 cells with the ethanol extract also caused dramatic increase in the PreG1 population with concomitant decrease in cycling cells, provoked DNA fragmentation, significant increase in the expression levels of p53 and Bax proteins and activated pro-apoptotic caspase-3. The results obtained suggest that the ethanol extract of AHE could be used as an easily accessible source of natural antioxidants and as potential phytochemicals against colon cancer.</p

Retinal pigment epithelial cells can be cultured on fluocinolone acetonide treated nanofibrous scaffold

Engineered tissue currently lacks requisite capacity to sustain cell viability and functionality. Here we demonstrate that human RPE cell lines (ARPE-19) can be cultured on ultrathin suspended electrospun nanofibre scaffolds (ENS) composed of hydrophobic polymer polyacrylonitrile (PAN) and a water-soluble aliphatic diamine, without (untreated) or with (treated) fluocinolone acetonide (FA). Cells survived and retained their characteristic morphology for up to 150 days with FA-treated ENS and manifested a morphological epithelial phenotype with expression of biomarkers critical for maintaining retinal physiological characteristics. This novel technique for producing culture substrates provides suitable hydrophilicity and a protective environment for prolonged RPE culture and has immense potential for subretinal transplantation. The findings indicate that FA-treated ENS is an excellent matrix for retaining the differentiated and epithelial phenotype.</p

Effects of <i>Smallanthus sonchifolius</i> Flour on Metabolic Parameters: A Systematic Review

Smallanthus sonchifolius, popularly known as yacon, is a member of the Asteraceae family. Due to its medicinal and edible value, yacon is consumed by different populations. Yacon is unique due to its high fructo-oligosaccharide and inulin content, as well as flavonoids, sesquiterpene lactones, and phenolic acids. Roots can be used to produce flour, which is less perishable and can be applied in various industrial products. This systematic review focuses on the effects of yacon flour on metabolic parameters. PubMed, Cochrane, Embase, Science Direct, Scopus, Web of Science, and Google Scholar databases were consulted, and PRISMA guidelines were followed in the selection of the studies. In total, 526 articles were found in the databases, and of these, only 28 full texts were eligible for inclusion. After applying the inclusion and exclusion criteria, seven studies were finally included. The results showed that the use of yacon flour can reduce glycemia, HbA1c, advanced glycation ends, plasma lipids, body fat mass, body weight, and waist circumference and improve intestinal microbiota and the antioxidant status. Further exploration of the effects of yacon flour is warranted, and additional clinical trials are necessary to determine the optimal daily consumption levels required to assist in improving metabolic parameters