TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Lević, Steva
AU  - Savić Šević, Svetlana
AU  - Pantelić, Dejan
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D
AU  - Krmpot, Aleksandar J
PY  - 2023
UR  - https://www.eventclass.org/contxt_emim2023/online-program/session?s=PW36#e609
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6289
AB  - Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.
PB  - European Society for Molecular Imaging
C3  - European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
T1  - Laser nano-surgery of fungal cell wall to enable patch clamping
SP  - 1095
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6289
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Lević, Steva and Savić Šević, Svetlana and Pantelić, Dejan and Živić, Miroslav and Rabasović, Mihailo D and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.",
publisher = "European Society for Molecular Imaging",
journal = "European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria",
title = "Laser nano-surgery of fungal cell wall to enable patch clamping",
pages = "1095",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6289"
}

Pajić, T., Stevanović, K., Todorović, N., Lević, S., Savić Šević, S., Pantelić, D., Živić, M., Rabasović, M. D.,& Krmpot, A. J.. (2023). Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
European Society for Molecular Imaging., 1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289

Pajić T, Stevanović K, Todorović N, Lević S, Savić Šević S, Pantelić D, Živić M, Rabasović MD, Krmpot AJ. Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria. 2023;:1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Lević, Steva, Savić Šević, Svetlana, Pantelić, Dejan, Živić, Miroslav, Rabasović, Mihailo D, Krmpot, Aleksandar J, "Laser nano-surgery of fungal cell wall to enable patch clamping" in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria (2023):1095,
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

TY  - JOUR
AU  - Kostić, Aleksandar Ž.
AU  - Milinčić, Danijel D.
AU  - Stanisavljević, Nemanja S.
AU  - Gašić, Uroš
AU  - Lević, Steva
AU  - Kojić, Milan O.
AU  - Lj Tešić, Živoslav
AU  - Nedović, Viktor
AU  - Barać, Miroljub B.
AU  - Pešić, Mirjana B.
PY  - 2021
UR  - https://linkinghub.elsevier.com/retrieve/pii/S0308814621003162
UR  - http://www.ncbi.nlm.nih.gov/pubmed/33640767
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4158
AB  - The aim of research was to determine polyphenols bioaccessibility and antioxidant properties of thermally-treated skimmed goat milk enriched with sunflower bee-collected pollen through in vitro digestion. HPLC analysis confirmed that pollen-enriched milk contained flavonols as the main phenolic fraction (80.7-76.2%) followed by phenolic acids (14.2-17.4%). Among individual compounds quercetin-3-O-glucoside (155.1-197.2 μg/L) and p-coumaric acid (29.5-30.7 μg/L) were the main quantified flavonols and phenolic acids, respectively. After digestion of milk/pollen sample, total polyphenols recovery was 30.71% with higher phenolic acids recovery (40.1%) compared to flavonols (28.3%) indicating strong interactions between caprine milk casein micelles and pollen polyphenols. Applied antioxidant assays (phosphomolybdenum, ABTS•+scavenging activity and ferrous-ion-chelating capacity) have confirmed complexity of prepared product- it had high ability to quench ABTS•+ radicals and to form chelating complexes with Fe2+ ions. Digestion provoked 20% reduction in total antioxidant capacity compared to the initial sample. TTSG milk/pollen powder could be good functional ingredient.
PB  - Elsevier
T2  - Food Chemistry
T1  - Polyphenol bioaccessibility and antioxidant properties of in vitro digested spray-dried thermally-treated skimmed goat milk enriched with pollen.
VL  - 351
DO  - 10.1016/j.foodchem.2021.129310
SP  - 129310
ER  - 

@article{
author = "Kostić, Aleksandar Ž. and Milinčić, Danijel D. and Stanisavljević, Nemanja S. and Gašić, Uroš and Lević, Steva and Kojić, Milan O. and Lj Tešić, Živoslav and Nedović, Viktor and Barać, Miroljub B. and Pešić, Mirjana B.",
year = "2021",
abstract = "The aim of research was to determine polyphenols bioaccessibility and antioxidant properties of thermally-treated skimmed goat milk enriched with sunflower bee-collected pollen through in vitro digestion. HPLC analysis confirmed that pollen-enriched milk contained flavonols as the main phenolic fraction (80.7-76.2%) followed by phenolic acids (14.2-17.4%). Among individual compounds quercetin-3-O-glucoside (155.1-197.2 μg/L) and p-coumaric acid (29.5-30.7 μg/L) were the main quantified flavonols and phenolic acids, respectively. After digestion of milk/pollen sample, total polyphenols recovery was 30.71% with higher phenolic acids recovery (40.1%) compared to flavonols (28.3%) indicating strong interactions between caprine milk casein micelles and pollen polyphenols. Applied antioxidant assays (phosphomolybdenum, ABTS•+scavenging activity and ferrous-ion-chelating capacity) have confirmed complexity of prepared product- it had high ability to quench ABTS•+ radicals and to form chelating complexes with Fe2+ ions. Digestion provoked 20% reduction in total antioxidant capacity compared to the initial sample. TTSG milk/pollen powder could be good functional ingredient.",
publisher = "Elsevier",
journal = "Food Chemistry",
title = "Polyphenol bioaccessibility and antioxidant properties of in vitro digested spray-dried thermally-treated skimmed goat milk enriched with pollen.",
volume = "351",
doi = "10.1016/j.foodchem.2021.129310",
pages = "129310"
}

Kostić, A. Ž., Milinčić, D. D., Stanisavljević, N. S., Gašić, U., Lević, S., Kojić, M. O., Lj Tešić, Ž., Nedović, V., Barać, M. B.,& Pešić, M. B.. (2021). Polyphenol bioaccessibility and antioxidant properties of in vitro digested spray-dried thermally-treated skimmed goat milk enriched with pollen.. in Food Chemistry
Elsevier., 351, 129310.
https://doi.org/10.1016/j.foodchem.2021.129310

Kostić AŽ, Milinčić DD, Stanisavljević NS, Gašić U, Lević S, Kojić MO, Lj Tešić Ž, Nedović V, Barać MB, Pešić MB. Polyphenol bioaccessibility and antioxidant properties of in vitro digested spray-dried thermally-treated skimmed goat milk enriched with pollen.. in Food Chemistry. 2021;351:129310.
doi:10.1016/j.foodchem.2021.129310 .

Kostić, Aleksandar Ž., Milinčić, Danijel D., Stanisavljević, Nemanja S., Gašić, Uroš, Lević, Steva, Kojić, Milan O., Lj Tešić, Živoslav, Nedović, Viktor, Barać, Miroljub B., Pešić, Mirjana B., "Polyphenol bioaccessibility and antioxidant properties of in vitro digested spray-dried thermally-treated skimmed goat milk enriched with pollen." in Food Chemistry, 351 (2021):129310,
https://doi.org/10.1016/j.foodchem.2021.129310 . .

TY  - JOUR
AU  - Milinčić, Danijel
AU  - Kostić, Aleksandar
AU  - Gašić, Uroš
AU  - Lević, Steva
AU  - Stanojević, Sladjana
AU  - Barać, Miroljub
AU  - Tešić, Živoslav
AU  - Nedović, Viktor
AU  - Pešić, Mirjana
PY  - 2021
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4316
AB  - The aim of this research was phenolics and protein characterization and antioxidant properties evaluation of skimmed thermally treated goat’s milk powder enriched with different concentration of grape pomace seed extract (SE). The dominant phenolics in SE were phenolic acids, flavan–3-ols and procyanidins. Different electrophoretic techniques together with UHPLC-MS/MS analysis revealed the presence of phenolics-protein interactions in the samples, mainly procyanidins with whey protein/caseins complexes. Addition of SE into thermally treated goat’s milk significantly improved antioxidant properties of goat’s milk such as TAC, FRP, DPPH• and ABTS•+ scavenging activity. Gallic acid, catechin, and procyanidins mostly contributed to these activities. The schematic representation of phenolics–casein micelles interactions in thermally treated goat’s milk enriched with SE was given. The addition of SE into thermally treated goat’s milk can be a promising strategy in food waste recovery and to enhance the beneficial health effects of goat’s milk-based functional foods.
PB  - Basel: MDPI
T2  - Biomolecules
T1  - Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties
IS  - 7
VL  - 11
DO  - 10.3390/biom11070965
SP  - 965
ER  - 

@article{
author = "Milinčić, Danijel and Kostić, Aleksandar and Gašić, Uroš and Lević, Steva and Stanojević, Sladjana and Barać, Miroljub and Tešić, Živoslav and Nedović, Viktor and Pešić, Mirjana",
year = "2021",
abstract = "The aim of this research was phenolics and protein characterization and antioxidant properties evaluation of skimmed thermally treated goat’s milk powder enriched with different concentration of grape pomace seed extract (SE). The dominant phenolics in SE were phenolic acids, flavan–3-ols and procyanidins. Different electrophoretic techniques together with UHPLC-MS/MS analysis revealed the presence of phenolics-protein interactions in the samples, mainly procyanidins with whey protein/caseins complexes. Addition of SE into thermally treated goat’s milk significantly improved antioxidant properties of goat’s milk such as TAC, FRP, DPPH• and ABTS•+ scavenging activity. Gallic acid, catechin, and procyanidins mostly contributed to these activities. The schematic representation of phenolics–casein micelles interactions in thermally treated goat’s milk enriched with SE was given. The addition of SE into thermally treated goat’s milk can be a promising strategy in food waste recovery and to enhance the beneficial health effects of goat’s milk-based functional foods.",
publisher = "Basel: MDPI",
journal = "Biomolecules",
title = "Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties",
number = "7",
volume = "11",
doi = "10.3390/biom11070965",
pages = "965"
}

Milinčić, D., Kostić, A., Gašić, U., Lević, S., Stanojević, S., Barać, M., Tešić, Ž., Nedović, V.,& Pešić, M.. (2021). Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties. in Biomolecules
Basel: MDPI., 11(7), 965.
https://doi.org/10.3390/biom11070965

Milinčić D, Kostić A, Gašić U, Lević S, Stanojević S, Barać M, Tešić Ž, Nedović V, Pešić M. Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties. in Biomolecules. 2021;11(7):965.
doi:10.3390/biom11070965 .

Milinčić, Danijel, Kostić, Aleksandar, Gašić, Uroš, Lević, Steva, Stanojević, Sladjana, Barać, Miroljub, Tešić, Živoslav, Nedović, Viktor, Pešić, Mirjana, "Skimmed Goat’s Milk Powder Enriched with Grape Pomace Seed Extract: Phenolics and Protein Characterization and Antioxidant Properties" in Biomolecules, 11, no. 7 (2021):965,
https://doi.org/10.3390/biom11070965 . .

TY  - JOUR
AU  - Stanojković, Jelena
AU  - Todorović, Slađana
AU  - Pećinar, Ilinka
AU  - Lević, Steva
AU  - Ćalić, Snežana
AU  - Janošević, Dušica
PY  - 2020
UR  - https://linkinghub.elsevier.com/retrieve/pii/S0926669020310189
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4071
AB  - Glandular trichomes are secretory organs that vary greatly in size, shape, location, type, and composition of secondary metabolites that are synthesized in them. They represent a protective chemical barrier and metabolic factories for the synthesis of bioactive compounds. We evaluated the effect of sucrose on the growth and biomass production, as well as on the distribution, density, and chemical composition of leaf glandular trichomes developed in vitro cultured Inula britannica, an important medical and aromatic species. Nodal segments (5 mm) were cultivated on the basal Murashige and Skoog medium with different concentrations (0 M, 0.01 M, 0.06 M, 0.1 M and 0.3 M) of sucrose. The lack of sugar does not retard growth, while a high sugar concentration inhibits biomass accumulation in vitro. The density and number of leaf glandular trichomes are changed with the medium composition variation. Morphoanatomical and histochemical analyses of the trichomes of the cultured I. britannica applying SEM, conventional light, fluorescent and Raman microscopy revealed the presence of non- glandular and biseriate glandular trichomes on the leaf surface. The histochemical analysis proved that glandular trichomes synthesized a complex mixture of biomolecules. The Raman microscopy analysis of glandular trichomes confirmed the presence of terpenes, most probably the bicyclic monoterpenes and sesquiterpenes. The multivariate analysis, based on PCA, was applied to distinguish among the glandular trichomes of three sugar concentrations: sugar-free medium (0 M), optimal (0.1 M) and the highest sucrose concentration (0.3 M). The main differences between the chemical profile of the leaf glandular trichomes of the plants grown on the 0 M and the chemical profile of the trichomes developed on the 0.1 and 0.3 M medium come mainly from phenolic compounds and to a smaller degree from cyclic terpenes, while the chemical profile of the glandular trichomes grown on the 0.1 M and 0.3 M differs considerably from the trichomes grown on the sugar-free medium in the polysaccharide cell wall ingredients. Most differences between the chemical profile of the glandular trichomes developed on 0 M and 0.3 M and the chemical profile of the trichomes grown on the 0.1 M of sucrose, come from phenolic compounds.
PB  - Elsevier BV
T2  - Industrial Crops and Products
T1  - Leaf glandular trichomes of micropropagated Inula britannica – Effect of sucrose on trichome density, distribution and chemical profile
DO  - 10.1016/j.indcrop.2020.113101
SP  - 113101
ER  - 

@article{
author = "Stanojković, Jelena and Todorović, Slađana and Pećinar, Ilinka and Lević, Steva and Ćalić, Snežana and Janošević, Dušica",
year = "2020",
abstract = "Glandular trichomes are secretory organs that vary greatly in size, shape, location, type, and composition of secondary metabolites that are synthesized in them. They represent a protective chemical barrier and metabolic factories for the synthesis of bioactive compounds. We evaluated the effect of sucrose on the growth and biomass production, as well as on the distribution, density, and chemical composition of leaf glandular trichomes developed in vitro cultured Inula britannica, an important medical and aromatic species. Nodal segments (5 mm) were cultivated on the basal Murashige and Skoog medium with different concentrations (0 M, 0.01 M, 0.06 M, 0.1 M and 0.3 M) of sucrose. The lack of sugar does not retard growth, while a high sugar concentration inhibits biomass accumulation in vitro. The density and number of leaf glandular trichomes are changed with the medium composition variation. Morphoanatomical and histochemical analyses of the trichomes of the cultured I. britannica applying SEM, conventional light, fluorescent and Raman microscopy revealed the presence of non- glandular and biseriate glandular trichomes on the leaf surface. The histochemical analysis proved that glandular trichomes synthesized a complex mixture of biomolecules. The Raman microscopy analysis of glandular trichomes confirmed the presence of terpenes, most probably the bicyclic monoterpenes and sesquiterpenes. The multivariate analysis, based on PCA, was applied to distinguish among the glandular trichomes of three sugar concentrations: sugar-free medium (0 M), optimal (0.1 M) and the highest sucrose concentration (0.3 M). The main differences between the chemical profile of the leaf glandular trichomes of the plants grown on the 0 M and the chemical profile of the trichomes developed on the 0.1 and 0.3 M medium come mainly from phenolic compounds and to a smaller degree from cyclic terpenes, while the chemical profile of the glandular trichomes grown on the 0.1 M and 0.3 M differs considerably from the trichomes grown on the sugar-free medium in the polysaccharide cell wall ingredients. Most differences between the chemical profile of the glandular trichomes developed on 0 M and 0.3 M and the chemical profile of the trichomes grown on the 0.1 M of sucrose, come from phenolic compounds.",
publisher = "Elsevier BV",
journal = "Industrial Crops and Products",
title = "Leaf glandular trichomes of micropropagated Inula britannica – Effect of sucrose on trichome density, distribution and chemical profile",
doi = "10.1016/j.indcrop.2020.113101",
pages = "113101"
}

Stanojković, J., Todorović, S., Pećinar, I., Lević, S., Ćalić, S.,& Janošević, D.. (2020). Leaf glandular trichomes of micropropagated Inula britannica – Effect of sucrose on trichome density, distribution and chemical profile. in Industrial Crops and Products
Elsevier BV., 113101.
https://doi.org/10.1016/j.indcrop.2020.113101

Stanojković J, Todorović S, Pećinar I, Lević S, Ćalić S, Janošević D. Leaf glandular trichomes of micropropagated Inula britannica – Effect of sucrose on trichome density, distribution and chemical profile. in Industrial Crops and Products. 2020;:113101.
doi:10.1016/j.indcrop.2020.113101 .

Stanojković, Jelena, Todorović, Slađana, Pećinar, Ilinka, Lević, Steva, Ćalić, Snežana, Janošević, Dušica, "Leaf glandular trichomes of micropropagated Inula britannica – Effect of sucrose on trichome density, distribution and chemical profile" in Industrial Crops and Products (2020):113101,
https://doi.org/10.1016/j.indcrop.2020.113101 . .