The Effect of Potassium Application on Sugar Beet (Beta vulgaris L.) under Salt Stress

Salt stress is an important type of abiotic stress that limits vegetative production in the world, particularly in arid and semi-arid climatic areas. The aim of this study is to mitigate salt stress damage in the sugar beet plant, which is an important part of crop production, with potassium application. An experiment was designed according to a design of random blocks with 4 different doses (10, 20, 40, 80 mg kg-1 K) of potassium and 3 different salt levels (0, 100, 150 mM NaCl) and 3 replicates. Leaf length, leaf width, fresh weight, malondialdehyde (MDA) content, membrane damage, relative water content was determined after harvest. The data obtained from the experiment were evaluated by one-way analysis of variance (One-Way ANOVA). According to the results of variance analysis, leaf width, leaf length, fresh weight, MDA content, membrane damage, relative water content were found to be statistically significant in salt x potassium interaction. Due to the positive effects of potassium on the parameters known to increase the plants%252339%253B stress tolerance, it is thought that it may be beneficial in reducing the salt stress in order to make the sugar beet less affected by salt stress

In vitro self-organized mouse small intestinal epithelial monolayer protocol

Developing protocols to obtain intestinal epithelial monolayers that recapitulate in vivo physiology to overcome the limitations of the organoids’ closed geometry has become of great interest during the last few years. Most of the developed culture models showed physiological-relevant cell composition but did not prove self-renewing capacities. Here, we show a simple method to obtain mouse small intestine-derived epithelial monolayers organized into proliferative crypt-like domains, containing stem cells, and differentiated villus-like regions, closely resembling the in vivo cell composition and distribution. In addition, we adapted our model to a tissue culture format compatible with functional studies and prove close to physiological barrier properties of our in vitro epithelial monolayers. Thus, we have set-up a protocol to generate physiologically relevant intestinal epithelial monolayers to be employed in assays where independent access to both luminal and basolateral compartments is needed, such as drug absorption, intracellular trafficking and microbiome-epithelium interaction assays

Imaging the cell-morphological response to 3D topography and curvature in engineered intestinal tissues

While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations

Potasyumun Domateste Kök-Ur Nematodu (Meloidogyne İncognita) Üzerine Etkisi

DergiPark: 791859tujesBu çalışmada; kök-ur nematodunun, domates bitkisi üzerindeki zararının potasyumla azaltılması amaçlanmıştır. Deneme bitki yetiştirme odasında; potasyumun 4 farklı dozu (10, 20, 40, 80 mg kg-1 K) ve 2 farklı nematod durumuyla (var, yok) 4 tekerrürlü olarak tesadüf blokları deneme desenine göre kurulmuştur. Bitkiler kum kültüründe, Hoagland besin solüsyonuyla yetiştirilmiştir. Bitkinin stresten etkilenme seviyesini değerlendirmek için hasattan sonra yaprak oransal su içeriği, membran zararlanması, klorofil a, klorofil b, toplam klorofil ve karotenoid içerikleri belirlenmiştir. Denemeden elde edilen veriler MINITAB 17.0 istatistik programıyla değerlendirilmiştir.Sonuçlara göre; köklerde ki urlanmalar, 0-10 ur skalasına göre düşük potasyum dozlarında (10, 20 mg kg-1 K) yüksek potasyum dozlarına (40, 80 mg kg-1 K) göre artmıştır. Potasyum uygulamaları ölçümleri yapılan parametreleri istatistiksel olarak etkilememiştir. Nematodun her iki (evet, hayır) durumunda da, potasyum dozu arttıkça, yaprak oransal su içeriği artarken, membran zararı azalmıştır. Elde edilen sonuçlara göre potasyum uygulamasının domates bitkilerinde kök-ur nematodunun (Meloidogyne incognita) verdiği zararı hafifletebileceği söylenebilmektedir

Dynamic photopolymerization produces complex microstructures on hydrogels in a moldless approach to generate a 3D intestinal tissue model

Epithelial tissues contain three-dimensional (3D) complex microtopographies that are essential for proper performance. These microstructures provide cells with the physicochemical cues needed to guide their self-organization into functional tissue structures. However, most in vitro models do not implement these 3D architectural features. The main problem is the availability of simple fabrication techniques that can reproduce the complex geometries found in native tissues on the soft polymeric materials required as cell culture substrates. In this study reaction-diffusion mediated photolithography is used to fabricate 3D microstructures with complex geometries on poly(ethylene glycol)-based hydrogels in a single step and moldless approach. By controlling fabrication parameters such as the oxygen diffusion/depletion timescales, the distance to the light source and the exposure dose, the dimensions and geometry of the microstructures can be well-defined. In addition, copolymerization of poly(ethylene glycol) with acrylic acid improves control of the dynamic reaction-diffusion processes that govern the free-radical polymerization of highly-diluted polymeric solutions. Moreover, acrylic acid allows adjusting the density of cell adhesive ligands while preserving the mechanical properties of the hydrogels. The method proposed is a simple, single-step, and cost-effective strategy for producing models of intestinal epithelium that can be easily integrated into standard cell culture platfor

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies

Modeling Biochemical Gradients In Vitro to Control Cell Compartmentalization in a Microengineered 3D Model of the Intestinal Epithelium

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH

Towards the development of biomimetic in vitro models of intestinal epithelium derived from intestinal organoids

[eng] Intestinal epithelium is highly specialized tissue organized into crypt-villus units relevant for their effective barrier function and nutrient absorption. In the crypt units reside the proliferative intestinal stem cells (ISCs) that divide and differentiate while migrating along the villi to generate the epithelium. The proliferation, migration and differentiation of ISCs is governed by the tightly controlled spatio-chemical gradients of ISC niche factors; bone morphogenic protein (BMP), wingless/Int (Wnt) and epidermal growth factor (EGF) pathway modulators. In vitro models of the intestinal epithelium, for the most part, based on culturing of intestinal stem cells/crypts in 3D cultures forming structures called organoids. These structures faithfully recapture diverse cell populations and their multicellular organization of native intestinal epithelium. However, 3D closed geometry of intestinal organoids prevents access to the apical region of the epithelium, making them unsuitable for conventional functionality assays. Experimental modeling of intestinal epithelial biology and physiology are limited due to the lack in vitro platforms that recapitulate these key aspects of the small intestinal epithelium: its distinct cell populations, 3D architecture and the gradients of ISC niche biochemical factors along the crypt-villus axis. Here, we describe development of in vitro models of intestinal epithelium obtained from intestinal organoid-derived crypts. First, we present a method that takes the advantage of substrate stiffness to dictate the formation of monolayers with accessible lumen rather than 3D organoids with a closed geometry. The 2D intestinal epithelium model has in vivo-like crypt-villus cellular organization with all major epithelial cell types and show physiologically relevant tissue barrier function. Then, we describe the development of a more complex model of intestinal epithelium by incorporating a 3D villus-like basement membrane substitute fabricated on hydrogels. For that, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are chosen due to their highly tunable chemical, and mechanical properties, porosity and photocrosslinkable nature allowing easy microstructuring. The formation of 3D bullet-like complex shapes was achieved by photolithography-based crosslinking of PEGDA, a simple, cost-effective approach. The bioactive functionalization of otherwise inert PEGDA for cell adhesion, was achieved by copolymerizing it with acrylic acid and a variety of cell adhesion proteins can be covalently anchored to the 3D villus-like hydrogels. We establish the optimal conditions for the growth of intestinal organoid-derived epithelial monolayers and demonstrated that organoid-derived intestinal epithelial cells successfully formed epithelial monolayers on collagen type I functionalized 3D villus-like PEGDA-acrylic acid hydrogels. Finally, we describe methods to create spatiotemporal gradients of biochemical ISC niche factors on 3D villus-like hydrogels and demonstrate that these gradients can be used to compartmentalize the differentiated epithelial cells. The spatio-chemical gradients of ISC niche biochemical factors on PEGDA hydrogels with proper porosity were successfully generated based on the free diffusion of the factors from a source to a sink chamber in a custom-made microfluidic device allocating the hydrogel and visualized with light-sheet fluorescence microscopy. In silico models were developed to simulate the spatio-chemical gradients formed within the hydrogels. The 3D villus-like PEGDA hydrogels were fabricated on porous membranes and successfully adapted to Transwell® inserts that permitted access to both sides of the hydrogel and the generation of spatio-chemical gradients. The gradients generated in this fashion can be used to compartmentalize the differentiated epithelial cells more towards the tips of the villus-like microstructures. The 3D villus-like platform improves the current models in providing cells with physiologically representative topographical and mechanical cues and biochemical gradients. Due to its utility, this platform might find uncountable applications. It can be used for the understanding of the basic biology of the intestinal epithelium. In addition, it can be used to culture human intestinal stem cells allowing for the screening of novel therapies and disease modeling.[spa] El epitelio intestinal es un tejido altamente especializado, organizado en unidades de criptas y vellosidades que son relevantes para sus eficaces funciones de barrera y absorción de nutrientes. En las unidades de criptas residen las células madre intestinales (ISC) proliferativas que se dividen y diferencian mientras migran a lo largo de las vellosidades, las cuales generan el epitelio maduro. En el epitelio maduro, las ISC y las células proliferativas se localizan en las criptas y las células absorbentes y secretoras diferenciadas en las vellosidades. La proliferación, migración y diferenciación de las ISC se rigen por los gradientes químicos espaciales altamente controlados de los factores de nicho de la ISC; Moduladores de la vía de bone morphogenic protein (BMP), wingless/Int (Wnt) y epidermal growth factor (EGF). El modelado experimental de la biología y la fisiología del epitelio intestinal está limitado debido a la falta de plataformas in vitro que recapitulan estos aspectos clave del epitelio del intestino delgado: sus distintas poblaciones celulares, la arquitectura 3D y los gradientes de factores bioquímicos de nicho ISC a lo largo del eje cripta-vellosidad. Aquí, describimos el desarrollo de modelos in vitro de epitelio intestinal obtenidos de criptas derivadas de organoides intestinales. En primer lugar, presentamos un método para obtener monocapas epiteliales intestinales 2D con lumen accesible y función de barrera fisiológica. A continuación, describimos el desarrollo de andamios biomiméticos 3D similares a vellosidades en hidrogeles de diacrilato de polietilenglicol (PEGDA) utilizando un enfoque fotolitográfico simple y rentable. Demostramos que nuestra plataforma de vellosidades sintéticas apoya la formación de monocapas epiteliales de células epiteliales intestinales derivadas de organoides. Finalmente, describimos métodos para crear gradientes espaciotemporales de factores nicho bioquímicos ISC en hidrogeles 3D similares a vellosidades y demostramos que estos gradientes se pueden usar para compartimentar las células epiteliales diferenciadas. La plataforma 3D que recrea las vellosidades intestinalesmejora los modelos actuales al proporcionar a las células las señales topográficas y mecánicas y los gradientes bioquímicos fisiológicamente representativos. Debido a su utilidad, esta plataforma puede encontrar innumerables aplicaciones. Puede ser utilizada para la comprensión de la biología básica del epitelio intestinal. Además, se puede utilizar para cultivar células madre intestinales humanas que permitan la detección de nuevas terapias y el modelado de enfermedades

Olası korneal stroma mühendisliği uygulamaları için çok katmanlı kaldırılabilir polietilenimin ve poli(L-lisin) filmleri.

In this study we fabricated free standing multilayer films of polyelectrolyte complexes for potential use in tissue engineering of corneal stroma by using the layer-by-layer (LbL) approach. In the formation of these LbL films negatively charged, photocrosslinkable (methacrylated) hyaluronic acid (MA-HA) was used along with polycations polyethyleneimine (PEI) and poly(L-lysine) (PLL). Type I collagen (Col) was blended in with PLL for improving the water absorption and cell attachment properties of the films. It was shown that the LbL films could be easily peeled off from glass substrates due to the photocrosslinking of one of the LbL components, the hyaluronic acid. Film growth and composition were monitored with FTIR-ATR. Heights of peaks at 3383 cm-1, and 2958 cm-1increased along with the bilayer number confirming the polymer build-up. Film integrity and thickness were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Films thicker than 5 bilayers (BLs) were found to be uniform in appearance and 10 BL (PEI/MeHA) films were calculated to be ca. 6 μm thick. Atomic force microscopy (AFM) revealed that as the number of BLs increased, surface roughness decreased. Activity of methacrylated hyaluronic acid was shown by the increased resistance of photocrosslinked multilayer films against hydrolysis by hyaluronidase. Patterns could be created on the films by photocrosslinking further proving that the crosslinking step is successful. Since the ultimate goal was to construct a corneal stroma PEI/MA-HA films were tested with corneal stroma cells, keratocytes. Cell proliferation on PEI/MA-HA films was quite poor in comparison to TCPS. In order to improve the cell adhesion the tests were repeated with PLL/MA-HA. Collagen was added to decrease the hydrophilicity and introduce cell adhesion sequences (Arg-Gly-Asp, RGD) to improve cell proliferation on the films and thus PLL+Col/MA-HA films were also tested. Introduction of collagen to the PLL/MA-HA films was found to decrease water retention of the multilayer films and improve cell viability and proliferation. Col+PLL/MA-HA LbL thus appear to be a promising platform for tissue engineering, especially of corneal stroma.M.S. - Master of Scienc