Tag Archives: life sciences

Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila

The female reproductive tract (female-RT) must decipher the repertoire of molecular cues received from the male during copulation in order to activate and coordinate tract functionality necessary for high fertility. In Drosophila, this modulation is partially driven by spermathecal secretory cells (SSC). The SSC are a layer of cuboidal secretory glandular cells surrounding the spermatheca capsule where sperm is stored. It is unclear, however, how the SSC regulate the system’s activity. *

In the article “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz show that mating activates the secretory machinery of the SSC.*

The SSC release a heterogeneous population of extracellular vesicles (EVs) which is involved in initiating and managing the increase in egg-laying, and possibly sperm storage. Moreover, sperm and male accessory gland proteins are essential for such mating-mediated SSC activity. Thus, mating regulates secretory/endocytic pathways required for trafficking of vesicles to SSC-female-RT target sites, which modulate and coordinate reproductive tract activity to achieve high fertility. *

The authors used atomic force microscopy to scan the extracellular vesicles (EVs).

The samples were scanned in liquid using AFM cantilever beam 3 (CB3) of the NANOSENSORS uniqprobe qp-BioAC-CI AFM probes. The qp-BioAC-CI AFM tips have been rounded to a nominal AFM tip radius of 30nm and are dedicated for cell imaging applications and measurements on soft and life science samples. The uniqprobe qp-BioAC-CI AFM probes feature three different rectangular AFM cantilevers on one side of the support chip and the cantilevers unite fairly high resonance frequencies with low force constants.

The reflective gold coating deposited on the detector side of the AFM cantilevers covers only the free end above where the AFM tip is located. Main advantages of the uniqprobe coating are considerably less AFM cantilever bending and reduced thermal drift particularly for measurements in liquid environments.

The samples were also scanned in QI mode, in which a force curve is measured on every pixel.*

From these force curves, the authors calculated the adhesion force and Young’s modulus. *

The profiles of adhesion and Young’s modulus were created by measuring the mean value of the particle in three different areas of 5 × 5 µm. The diameter (nm) was obtained by creating a cross section of each EV and measuring the base of the profile. *

The involvement of EVs in SSC-female-RT routes of communication has added another layer of complexity to the process driving the switch towards a functional female-RT, and to high fertility. The precise mechanism by which male-derived signals, including EVs, affect SSC-derived trafficking has yet to be resolved. Deciphering male-female communication via EVs in Drosophila and other organisms will contribute greatly to our understanding of the different combinations of input modalities and output networks leading to high fertility. *

Fig. 5 from “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez et al.: The spermatheca releases EVs with specific characteristics. a–e The spermathecae (SSC > CD63-GFP) were cultured ex vivo to characterize the particles that were released to the spent media: a size distribution of CD63-GFP-positive particles (n = 25 spermathecae from 15 flies) using single-particle tracking of the Nanoimager. b Characterization of CD63-GFP particles by ExoView (100 spermathecae from 50-60 flies) using anti-GFP or anti-CD63. c Representative dSTORM image of EVs found in the spent media of SSC expressing CD63-GFP (n = 25 spermathecae from 15 flies), stained with AlexaFluor555 conjugated anti-CD63 primary antibody. The image shows CD63-GFP-positive EVs and single CD63 AlexaFluor555 molecules the EV’s surface. Scale bars = 1 µm and insets = 20 nm. d–e The morphology of EVs in the spent media was observed by e negative staining in STEM; scale bar = 200 nm; and by d Cryo-TEM; Scale bar = 100 nm (see also Supplementary Fig. 5a-c). f–i The spent media of ex vivo cultured spermathecae were analyzed for the presence of EVs: f Representative AFM scans of SSC-EVs isolated by acoustic sorting (n = 100 spermathecae from 70 flies), showing from left to right: height (nm), adhesion (pN) and Young’s modulus images (MPa). Scale bars = 100 nm (see Supplementary Fig. 5d–g). g–i Single-particle tracking in the nanoimager. g CellMaskTM-positive particle size distribution and concentration profiles of EVs in the spent media of spermathecae incubated in media alone (No ecdy) or with ecdysone (Ecdy) (see also Supplementary Movie 5 for a time lapse of spermathecae end apparatus and endosomal activity post-ecdysone stimulation and methods, ex vivo spermatheca culture section); media with only CellMaskTM stain served as a control for the formation of dye aggregates. h Particle concentration and i mean particle diameter (nm) from g; Box plots are the measurements of particles from four frames of spent media from 25 spermathecae; boxes represent maximum, median and minimum values with outliers; one-way ANOVA, with nonparametric Wilcoxon multiple comparison post-hoc test; *p < 0.0001. NANOSENSORS uniqprobe qp-BioAC-CI AFM probes (CB3) were used for the Atomic Force Microscopy in liquid
Fig. 5 from “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez et al.:
The spermatheca releases EVs with specific characteristics.
a–e The spermathecae (SSC > CD63-GFP) were cultured ex vivo to characterize the particles that were released to the spent media: a size distribution of CD63-GFP-positive particles (n = 25 spermathecae from 15 flies) using single-particle tracking of the Nanoimager. b Characterization of CD63-GFP particles by ExoView (100 spermathecae from 50-60 flies) using anti-GFP or anti-CD63. c Representative dSTORM image of EVs found in the spent media of SSC expressing CD63-GFP (n = 25 spermathecae from 15 flies), stained with AlexaFluor555 conjugated anti-CD63 primary antibody. The image shows CD63-GFP-positive EVs and single CD63 AlexaFluor555 molecules the EV’s surface. Scale bars = 1 µm and insets = 20 nm. d–e The morphology of EVs in the spent media was observed by e negative staining in STEM; scale bar = 200 nm; and by d Cryo-TEM; Scale bar = 100 nm (see also Supplementary Fig. 5a-c). f–i The spent media of ex vivo cultured spermathecae were analyzed for the presence of EVs: f Representative AFM scans of SSC-EVs isolated by acoustic sorting (n = 100 spermathecae from 70 flies), showing from left to right: height (nm), adhesion (pN) and Young’s modulus images (MPa). Scale bars = 100 nm (see Supplementary Fig. 5d–g). g–i Single-particle tracking in the nanoimager. g CellMaskTM-positive particle size distribution and concentration profiles of EVs in the spent media of spermathecae incubated in media alone (No ecdy) or with ecdysone (Ecdy) (see also Supplementary Movie 5 for a time lapse of spermathecae end apparatus and endosomal activity post-ecdysone stimulation and methods, ex vivo spermatheca culture section); media with only CellMaskTM stain served as a control for the formation of dye aggregates. h Particle concentration and i mean particle diameter (nm) from g; Box plots are the measurements of particles from four frames of spent media from 25 spermathecae; boxes represent maximum, median and minimum values with outliers; one-way ANOVA, with nonparametric Wilcoxon multiple comparison post-hoc test; *p < 0.0001.

*Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz
Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila
Nature Communications Biology volume 5, Article number: 815 (2022)
DOI: https://doi.org/10.1038/s42003-022-03770-6

Please follow this external link to read the full article: https://rdcu.be/cYtUq

Open Access: The article “Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila” by Javier Arturo Sanchez-Lopez, Shai Twena, Ido Apel, Shani Chen Kornhaeuser, Michael Chasnitsky, Andras G. Miklosi, Perla J. Vega-Dominguez, Alex Shephard, Amir Hefetz and Yael Heifetz is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.

Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces

The human skin is the second largest organ of the human body. The stratum corneum (SC) is the outmost layer of the human skin and performs various protective and adaptive physiological functions such as protecting against physical, chemical and biological damages.*

The homeostasis of the lipid matrix of the stratum corneum is essential for the correct functioning of the SC. If the composition of this lipid matrix is changed or disturbed skin ailments such as atopic dermatitis or psoriasis can be the result. According to various studies direct replenishment of the SC lipids on damaged skin has positive effects on the recovery of its barrier properties.*

Cerosomes or stratum corneum liposomes are a relatively new class of liposomes which are being investigated for the application as skin barrier repairing agents in chronical skin diseases.*

In the article “Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces” Fabio Strati, Tetiana Mukhina, Reinhard H. H. Neubert, Lukas Opalka, Gerd Hause, Christian E. H. Schmelzer, Matthias Menzel, Gerald Brezesinski describe how they prepared cerosomes, i.E. liposomes composed of SC lipids in order to investigate the mechanism of interaction with a 2D model of the SC lipid matrix.*

After a first step of characterizing the used SC model monolayer in detail they carried out the development of stable SC liposomes, the so-called cerosomes, in a second step.*

Once the cerosome formulations were developed and characterized, the interaction between these and monolayers of the SC lipid matrix model was investigated.*

The interaction was probed by means of adsorption isotherms after subphase injection, and after the transfer to a solid support by atomic force microscopy (AFM) measurements.*

The AFM experiments were performed to gain information about the structures of the formed assemblies. This technique allows to resolve the lateral organization and to visualize the presence of lipid domains and/or adsorbed vesicles be performing topographic surface measurements of the sample deposited onto a solid support with an Angstrom resolution in transversal direction.*

Topographical images were recorded in liquid state using NANOSENSORS uniqprobe qp-BioT AFM probes in a standard liquid cell containing the needed buffer.*

The results obtained with the application of AFM showed that the liposomes were able to both penetrate into empty spaces and lower domains present in the SC model monolayer and get adsorbed at the monolayer forming localized multilayers.*

The results presented in the article indicate that a strong interaction occurred between SC monolayers and the cerosomes.*

The study proves for the first time the mode of action by which cerosomes exploit their function as skin barrier repairing agents on the SC.*

The use of such formulations might not only be limited to restore the damaged skin but they could be also used to deliver active pharmaceutical ingredients encapsulated in the cerosomes. This might open new and interesting scenarios for treating skin conditions such as inflammations caused by atopic dermatitis and/or psoriasis.*

Fig. 7 from “Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces” by F. Strati et al: AFM scans of a) SC model monolayer transferred via LB method onto mica support, b) SC model monolayer after injection of cerosomes, c) SC model monolayer after injection of cerosome + S75-3 formulation, and d) SC model monolayer after injection of S75-3 liposomal formulation. All samples were transferred via the LS method onto glass substrate. Each experiment was performed at 20°C and the subphase used for a) was Millipore water while for b), c), and d) the same aqueous solutions have been used as for the formulation of the liposomes. Topographical images were recorded in liquid state using NANOSENSORS uniqprobe qp-BioT AFM probes in a standard liquid cell containing the needed buffer.
Fig. 7 from “Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces” by F. Strati et al: AFM scans of a) SC model monolayer transferred via LB method onto mica support, b) SC model monolayer after injection of cerosomes, c) SC model monolayer after injection of cerosome + S75-3 formulation, and d) SC model monolayer after injection of S75-3 liposomal formulation. All samples were transferred via the LS method onto glass substrate. Each experiment was performed at 20°C and the subphase used for a) was Millipore water while for b), c), and d) the same aqueous solutions have been used as for the formulation of the liposomes.

 

*Fabio Strati, Tetiana Mukhina, Reinhard H. H. Neubert, Lukas Opalka, Gerd Hause, Christian E. H. Schmelzer, Matthias Menzel, Gerald Brezesinski
Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces
BBA Advances, Volume 2, 2022, 100039
DOI: https://doi.org/10.1016/j.bbadva.2021.100039

Please follow this external link to read the full article:  https://doi.org/10.1016/j.bbadva.2021.100039

Open Access: The article “Cerosomes as skin repairing agent: Mode of action studies with a model stratum corneum layer at liquid/air and liquid/solid interfaces” by Fabio Strati, Tetiana Mukhina, Reinhard H. H. Neubert, Lukas Opalka, Gerd Hause, Christian E. H. Schmelzer, Matthias Menzel, Gerald Brezesinski is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds

Biocompatible scaffolds that can be repopulated with human cells have many uses such serving as replacement organs and tissues. Therefore there is an increasing interest in plant-based biomaterials for tissue engineering.*

As the above mentioned scaffolds should mimic the in vivo tissue environment closely they need to provide a fitting structural and biomechanical support to the cells while at the same time promoting cell behaviour and tissue development. *

Currently the standard method to prepare plant tissue to serve as a biocompatible scaffold is to decellularize it with serial chemical treatment.*

In their article “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds” Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern explore another method to produce biocompatible scaffolds.*

They use supercritical carbon dioxide (scCO2) together with 2% peracetic acid to decellularize plant material.*

Their subsequent investigations show that the process of decellularization, scaffold structure preservation and recellularization with human cells is less time consuming than with the standard chemical method.

In a further step the authors of the article describe how they use various scientific methods to evaluate the scaffolds they decellularized by the described scCO2 method.*

Ashlee F. Harris et al. use Atomic Force Microscopy (AFM) in order to find out if the scCO2 treatment had an impact on the mechanical properties of the scaffolds produced with this method.*

With AFM topography measurements they are able to establish that structures such as plant vasculature were preserved.*

The following determination of the Young’s Modulus calculated from multiple force curves of a homogeneous surface section of the produced scaffold shows it to be slightly lower than the one from a chemically decellularized scaffold.*

NANOSENSORS™ uniqprobe qp-BioAC AFM probes ( CB3 nominal values: 80 μm length, 30 μm mean width, 400 nm thickness, force constant 0.06 N/m, resonance frequency 30 kHz) were used for the scaffold measurements with Atomic Force Microscopy.

Figure 3 from “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds AFM imaging and spectrometry measurement” by Ashlee F. Harris et al.: 
They used AFM surface topography measurements to confirm that the structures such as plant vasculature were preserved after the scSO2 process and used  AFM force curves to calculate the  Young’s Modulus (YM) of the scCO2 decellularized scaffold. NANOSENSORS uniqprobe qp-BioAC AFM probes were used for the described AFM measurments. 
(a) Representative false colored three-dimensional surface mapping images and (b) Young’s modulus of scCO2 and chemically decellularized scaffolds (data as mean ± SEM; n = 5).
Figure 3 from “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds AFM imaging and spectrometry measurement” by Ashlee F. Harris et al.: (a) Representative false colored three-dimensional surface mapping images and (b) Young’s modulus of scCO2 and chemically decellularized scaffolds (data as mean ± SEM; n = 5).

While the scCo2 method promises to be a faster way to decellularize plant material and produce sterile and biocompatible scaffolds further research will be necessary to determine whether the differences the authors detected between the scaffolds produced with the scCO2 approach and the scaffolds produced with the chemical approach have a major influence on how repopulated cells behave in the achieved scaffolds.*

*Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern
Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds
Nature Scientific Reports 11, 3643 (2021)
DOI: https://doi.org/10.1038/s41598-021-83250-9

Please follow this external link to read the full article: https://rdcu.be/cAqW3

Open Access The article “Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds” by Ashlee F. Harris, Jerome Lacombe, Sumedha Liyanage, Margaret Y. Han, Emily Wallace, Sophia Karsunky, Noureddine Abidi and Frederic Zenhausern is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.