A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.

3D bioprinted endometrial stem cells on melt electrospun poly ε-caprolactone mesh for pelvic floor application promote anti-inflammatory responses in mice.

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports an alternative treatment strategy targeting vaginal wall repair using bioprinting of eMSCs encapsulated in a hydrogel and 3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly ε-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100 °C and with a speed 20 mm/s had the largest open pore diameter (47.2 ±â€¯11.4 µm) and the lowest strand thickness (121.4 ±â€¯46 µm) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of 3D bioprinted eMSC-MES meshes as an alternative approach to overcome the current challenges with non-degradable knitted meshes in POP treatment. STATEMENT OF SIGNIFICANCE: This study presents the first report of bioprinting mesenchymal stem cells derived from woman endometrium (eMSCs) to boost Pelvic Organ Prolapse (POP) treatment. It impacts over 50% of elderly women with no optimal treatment at present. The overall study is conducted in three stages as fabricating a melt electrospun (MES) mesh, bioprinting eMSCs into a Ca2+ free Aloe Vera-Alginate (AV-Alg) based hydrogel and in vivo study. Our data showed that AV-ALG hydrogel potentially suppresses the foreign body response and further addition of eMSCs triggered a high influx of anti-inflammatory CD206+ M2 macrophages. Our final construct demonstrates a favourable foreign body response to predict expected tissue integration, therefore, provides a potential for developing an alternative treatment for POP.

Targeted drug delivery using genetically engineered diatom biosilica.

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.

Surface-initiated hyperbranched polyglycerol as an ultralow-fouling coating on glass, silicon, and porous silicon substrates.

Anionic ring-opening polymerization of glycidol was initiated from activated glass, silicon, and porous silicon substrates to yield thin, ultralow-fouling hyperbranched polyglycerol (HPG) graft polymer coatings. Substrates were activated by deprotonation of surface-bound silanol functionalities. HPG polymerization was initiated upon the addition of freshly distilled glycidol to yield films in the nanometer thickness range. X-ray photoelectron spectroscopy, contact angle measurements, and ellipsometry were used to characterize the resulting coatings. The antifouling properties of HPG-coated surfaces were evaluated in terms of protein adsorption and the attachment of mammalian cells. The adsorption of bovine serum albumin and collagen type I was found to be reduced by as much as 97 and 91%, respectively, in comparison to untreated surfaces. Human glioblastoma and mouse fibroblast attachment was reduced by 99 and 98%, respectively. HPG-grafted substrates outperformed polyethylene glycol (PEG) grafted substrates of comparable thickness under the same incubation conditions. Our results demonstrate the effectiveness of antifouling HPG graft polymer coatings on a selected range of substrate materials and open the door for their use in biomedical applications.