TY  - JOUR
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J
AU  - Živić, Miroslav
AU  - Savić-Šević, Svetlana
AU  - Lević, Steva M
AU  - Stanić, Marina
AU  - Pantelić, Dejan
AU  - Jelenković, Brana
AU  - Rabasović, Mihailo D
PY  - 2024
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6666
AB  - Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.
PB  - Springer Nature
T2  - Microsystems & Nanoengineering
T1  - In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi
VL  - 10
DO  - 10.1038/s41378-024-00664-x
SP  - 47
ER  - 

@article{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar J and Živić, Miroslav and Savić-Šević, Svetlana and Lević, Steva M and Stanić, Marina and Pantelić, Dejan and Jelenković, Brana and Rabasović, Mihailo D",
year = "2024",
abstract = "Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.",
publisher = "Springer Nature",
journal = "Microsystems & Nanoengineering",
title = "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi",
volume = "10",
doi = "10.1038/s41378-024-00664-x",
pages = "47"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A. J., Živić, M., Savić-Šević, S., Lević, S. M., Stanić, M., Pantelić, D., Jelenković, B.,& Rabasović, M. D.. (2024). In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering
Springer Nature., 10, 47.
https://doi.org/10.1038/s41378-024-00664-x

Pajić T, Stevanović K, Todorović N, Krmpot AJ, Živić M, Savić-Šević S, Lević SM, Stanić M, Pantelić D, Jelenković B, Rabasović MD. In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering. 2024;10:47.
doi:10.1038/s41378-024-00664-x .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar J, Živić, Miroslav, Savić-Šević, Svetlana, Lević, Steva M, Stanić, Marina, Pantelić, Dejan, Jelenković, Brana, Rabasović, Mihailo D, "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi" in Microsystems & Nanoengineering, 10 (2024):47,
https://doi.org/10.1038/s41378-024-00664-x . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Nikolić, Stanko N
AU  - Rabasović, Mihailo D
AU  - Clayton, Andrew HA
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/123456789/6288
AB  - Introduction
Studies of lipid droplet (LD) physiology in fungi are still in their infancy but their quantitation
has relevance to issues in biomedicine, agriculture and industrial waste. Third Harmonic
Generation (THG) microscopy is non-invasive, produces inherently confocal images and
doesn’t require fixation or external labeling, which make it suitable for in vivo LD imaging [1,
2]. We present in vivo and label-free imaging of LD in individual fungal cells by THG
microscopy to assess the effects of nitrogen starvation. The LD quantification was performed
by two image analysis techniques.
Methods
THG microscopy was applied for the first time to a filamentous fungus and our choice was the
oleaginous fungus Phycomyces blakesleeanus. To observe the changes in LD number, the
22h old hyphae culture was divided into control and nitrogen starved groups (N-starved). A
home built nonlienar microscope with Yb:KGW laser at 1040 nm (200 fs pulses, 83 MHz
repetition rate) was used for THG imaging of live unstained hyphae [3]. THG signal was
detected by PMT in the transmission arm after passing through a Hoya glass UV filter with the
peak at 340 nm. 2D THG images of LDs (Fig. 1a) were analyzed by Image Correlation
Spectroscopy (ICS) measuring spatially-correlated fluctuations [4] and software particle
counting – Particle Size Analysis (PSA).
Results/Discussion
The small volume of hyphae suspension was placed between two coverslips of 170 μm
thickness in order to meet the criteria for the best numerical aperture of the objective lens and
for better transmission of THG signal. The high resolution of the microscopic system, the
hyphae thickness (ca 10 μm) and medium transparency made it possible for the whole
hyphae to be optically sectioned and a 3D model to be reconstructed (Fig. 1b and video).
Since ICS was primarily developed for fluorescent images and was not used to analyze THG
images, we have tested it by comparing the results to the PSA. Nitrogen starvation as
expected [5] increased LD number compared to control which was confirmed by both methods and obtained results are in good agreement. The overall increase of LDs during
growth without available nitrogen is found to be between 3 and 4.5 h time point, followed with
the loss of population of larger-than-average LDs during prolonged starvation.
Conclusions
THG microscopy is suitable for imaging and quantification of changes in lipid droplet number,
brought upon by complete removal of nitrogen, from such low density/diameter baseline. In
addition, we demonstrate that the ICA is suitable for THG images, although it is primarily
developed and have been mostly used for fluorescence signals so far.
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  - Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation
SP  - 1093
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6288
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Nikolić, Stanko N and Rabasović, Mihailo D and Clayton, Andrew HA and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Studies of lipid droplet (LD) physiology in fungi are still in their infancy but their quantitation
has relevance to issues in biomedicine, agriculture and industrial waste. Third Harmonic
Generation (THG) microscopy is non-invasive, produces inherently confocal images and
doesn’t require fixation or external labeling, which make it suitable for in vivo LD imaging [1,
2]. We present in vivo and label-free imaging of LD in individual fungal cells by THG
microscopy to assess the effects of nitrogen starvation. The LD quantification was performed
by two image analysis techniques.
Methods
THG microscopy was applied for the first time to a filamentous fungus and our choice was the
oleaginous fungus Phycomyces blakesleeanus. To observe the changes in LD number, the
22h old hyphae culture was divided into control and nitrogen starved groups (N-starved). A
home built nonlienar microscope with Yb:KGW laser at 1040 nm (200 fs pulses, 83 MHz
repetition rate) was used for THG imaging of live unstained hyphae [3]. THG signal was
detected by PMT in the transmission arm after passing through a Hoya glass UV filter with the
peak at 340 nm. 2D THG images of LDs (Fig. 1a) were analyzed by Image Correlation
Spectroscopy (ICS) measuring spatially-correlated fluctuations [4] and software particle
counting – Particle Size Analysis (PSA).
Results/Discussion
The small volume of hyphae suspension was placed between two coverslips of 170 μm
thickness in order to meet the criteria for the best numerical aperture of the objective lens and
for better transmission of THG signal. The high resolution of the microscopic system, the
hyphae thickness (ca 10 μm) and medium transparency made it possible for the whole
hyphae to be optically sectioned and a 3D model to be reconstructed (Fig. 1b and video).
Since ICS was primarily developed for fluorescent images and was not used to analyze THG
images, we have tested it by comparing the results to the PSA. Nitrogen starvation as
expected [5] increased LD number compared to control which was confirmed by both methods and obtained results are in good agreement. The overall increase of LDs during
growth without available nitrogen is found to be between 3 and 4.5 h time point, followed with
the loss of population of larger-than-average LDs during prolonged starvation.
Conclusions
THG microscopy is suitable for imaging and quantification of changes in lipid droplet number,
brought upon by complete removal of nitrogen, from such low density/diameter baseline. In
addition, we demonstrate that the ICA is suitable for THG images, although it is primarily
developed and have been mostly used for fluorescence signals so far.",
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 = "Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation",
pages = "1093",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6288"
}

Pajić, T., Todorović, N., Živić, M., Nikolić, S. N., Rabasović, M. D., Clayton, A. H.,& Krmpot, A. J.. (2023). Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation. 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., 1093.
https://hdl.handle.net/21.15107/rcub_ibiss_6288

Pajić T, Todorović N, Živić M, Nikolić SN, Rabasović MD, Clayton AH, Krmpot AJ. Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria. 2023;:1093.
https://hdl.handle.net/21.15107/rcub_ibiss_6288 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Nikolić, Stanko N, Rabasović, Mihailo D, Clayton, Andrew HA, Krmpot, Aleksandar J, "Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation" in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria (2023):1093,
https://hdl.handle.net/21.15107/rcub_ibiss_6288 .

TY  - CONF
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Cvetić Antić, Tijana
AU  - Atlagić, Kristina
AU  - Krmpot, Aleksandar
PY  - 2023
UR  - https://microbiology.bg.ac.rs/workshop2023/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6248
AB  - The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.
PB  - Belgrade: University of Belgrade, Faculty of Biology
C3  - Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
T1  - Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent
SP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6248
ER  - 

@conference{
author = "Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Cvetić Antić, Tijana and Atlagić, Kristina and Krmpot, Aleksandar",
year = "2023",
abstract = "The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia",
title = "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent",
pages = "84",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6248"
}

Todorović, N., Živić, M., Stevanović, K., Pajić, T., Cvetić Antić, T., Atlagić, K.,& Krmpot, A.. (2023). Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
Belgrade: University of Belgrade, Faculty of Biology., 84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248

Todorović N, Živić M, Stevanović K, Pajić T, Cvetić Antić T, Atlagić K, Krmpot A. Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia. 2023;:84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Cvetić Antić, Tijana, Atlagić, Kristina, Krmpot, Aleksandar, "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent" in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia (2023):84,
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Žižić, Milan
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6246
AB  - The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.
PB  - Kragujevac: Institute for Information Technologies, University of Belgrade
C3  - Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
T1  - Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts
DO  - 10.46793/ICCBI23.221S
SP  - 221
EP  - 224
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Krmpot, Aleksandar and Rabasović, Mihailo and Žižić, Milan and Živić, Miroslav and Todorović, Nataša",
year = "2023",
abstract = "The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.",
publisher = "Kragujevac: Institute for Information Technologies, University of Belgrade",
journal = "Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia",
title = "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts",
doi = "10.46793/ICCBI23.221S",
pages = "221-224"
}

Stevanović, K., Pajić, T., Krmpot, A., Rabasović, M., Žižić, M., Živić, M.,& Todorović, N.. (2023). Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
Kragujevac: Institute for Information Technologies, University of Belgrade., 221-224.
https://doi.org/10.46793/ICCBI23.221S

Stevanović K, Pajić T, Krmpot A, Rabasović M, Žižić M, Živić M, Todorović N. Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia. 2023;:221-224.
doi:10.46793/ICCBI23.221S .

Stevanović, Katarina, Pajić, Tanja, Krmpot, Aleksandar, Rabasović, Mihailo, Žižić, Milan, Živić, Miroslav, Todorović, Nataša, "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts" in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia (2023):221-224,
https://doi.org/10.46793/ICCBI23.221S . .

TY  - CONF
AU  - Vitkovac, Aleksandra
AU  - Pajić, Tanja
AU  - Bukumira, Marta
AU  - Stanić, Marina
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6247
AB  - Adaptive changes in mitochondrial morphology are associated with changes in the mitochondrial function and metabolic fitness of eukaryotic cells. We previously described in young hyphae of the filamentous fungus Phycomyces blakesleeanus a dramatic effect of an increase in ambient temperature during growth: a 3°C warmer environment compared with a control temperature of 22°C resulted in the appearance of long elongated (“tubular”) mitochondria accompanied by an increase in lipid droplet density. Here, we examined how cooler ambient temperature (18°C) during growth affects mitochondrial morphology in P. blakesleeanus compared with the control grown at 22°C. We used two-photon fluorescence imaging (TPEF) of live hyphae stained with the vital mitochondrial dye rhodamine 123. Extraction of relevant parameters (number, size, and shape of mitochondria) from TPEF images was performed using the Ilastik machine learning-based software. The suitability of the Ilastik analysis was compared with the Particle Analysis (ImageJ). Cold treatment resulted in the appearance of tubular mitochondrial morphology that was absent in the control group. Tubular mitochondrial morphology appears to be an adaptive feature that occurs in both warmer and colder conditions and is likely part of the stress response.
PB  - Kragujevac: Institute for Information Technologies, University of Belgrade
C3  - Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
T1  - Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology
DO  - 10.46793/ICCBI23.334V
SP  - 334
EP  - 337
ER  - 

@conference{
author = "Vitkovac, Aleksandra and Pajić, Tanja and Bukumira, Marta and Stanić, Marina and Rabasović, Mihailo and Todorović, Nataša",
year = "2023",
abstract = "Adaptive changes in mitochondrial morphology are associated with changes in the mitochondrial function and metabolic fitness of eukaryotic cells. We previously described in young hyphae of the filamentous fungus Phycomyces blakesleeanus a dramatic effect of an increase in ambient temperature during growth: a 3°C warmer environment compared with a control temperature of 22°C resulted in the appearance of long elongated (“tubular”) mitochondria accompanied by an increase in lipid droplet density. Here, we examined how cooler ambient temperature (18°C) during growth affects mitochondrial morphology in P. blakesleeanus compared with the control grown at 22°C. We used two-photon fluorescence imaging (TPEF) of live hyphae stained with the vital mitochondrial dye rhodamine 123. Extraction of relevant parameters (number, size, and shape of mitochondria) from TPEF images was performed using the Ilastik machine learning-based software. The suitability of the Ilastik analysis was compared with the Particle Analysis (ImageJ). Cold treatment resulted in the appearance of tubular mitochondrial morphology that was absent in the control group. Tubular mitochondrial morphology appears to be an adaptive feature that occurs in both warmer and colder conditions and is likely part of the stress response.",
publisher = "Kragujevac: Institute for Information Technologies, University of Belgrade",
journal = "Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia",
title = "Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology",
doi = "10.46793/ICCBI23.334V",
pages = "334-337"
}

Vitkovac, A., Pajić, T., Bukumira, M., Stanić, M., Rabasović, M.,& Todorović, N.. (2023). Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
Kragujevac: Institute for Information Technologies, University of Belgrade., 334-337.
https://doi.org/10.46793/ICCBI23.334V

Vitkovac A, Pajić T, Bukumira M, Stanić M, Rabasović M, Todorović N. Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia. 2023;:334-337.
doi:10.46793/ICCBI23.334V .

Vitkovac, Aleksandra, Pajić, Tanja, Bukumira, Marta, Stanić, Marina, Rabasović, Mihailo, Todorović, Nataša, "Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology" in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia (2023):334-337,
https://doi.org/10.46793/ICCBI23.334V . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Kozakijević, Suzana
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
AU  - Todorović, Nataša
AU  - Rabasović, Mihailo D.
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6249
AB  - Mitochondrial function, and consequently cellular metabolic status and fitness of a cell, is tightly linked
to the dynamic changes of mitochondrial morphology, including mitochondrial fusion, fission and
mitophagy [1]. Lipid droplets (LDs) can be in close contact with mitochondria, and accumulate
autophagy or mitophagy generated material during the reparatory processes [2]. The effect of increased
ambient temperature on mitochondrial morphology and LDs density in living cells of the filamentous
fungus Phycomyces blakesleeanus was investigated. For in vivo imaging of mitochondria and LDs
multiphoton microscopy was used. Multiphoton microscopy enables 3D imaging in high resolution and
reduced photodamage and photobleaching of the sample using IR ultrafast pulsed lasers. Mitochondria
were stained with the vital dye Rhodamine123 (Rh123) which enters these organelles based on their
membrane potential - mitochondria must be healthy/active to stain. A wavelength of 800 nm from Ti:Sa
laser (160 fs pulse duration, 76 MHz repetition rate) was used for two-photon excitation of Rh123. The
laser beam was focused by the Zeiss Plan Neofluar 40x1.3 objective lens and the signal was detected
through a bandpass interference filter MF530/43 (ThorLabs, USA). For LDs staining a Nile Red dye
was used and excited by Yb: KGW laser at 1040 nm [3]. Six morphological types of mitochondria were
observed in the hyphae of this fungus: intermediate type - normal, intermediate with small semicircular
tubules, tubular, elongated tubular, fragmented (small ellipsoid tubule) and fragmented with exclusively
spheroid-shaped mitochondria. Changes in mitochondrial morphology were induced by a small
temperature change. A 3°C increase in ambient temperature, from 22°C, had a dramatic effect on
mitochondrial morphology, inducing the appearance of a predominantly tubular mitochondrial
morphology. The total area percentage of mitochondria showed an increasing trend when grown at
25°C. Increasing the ambient temperature to 25°C induced a statistically significant increase in the
percentage of hyphal area occupied by LDs from 2.9 ± 1.6 to 4.7 ± 2.2 (mean value and SD in percentage
of hyphal area). The observed response to the small temperature increase points to the physiological
adaptation of hyphal metabolism.
PB  - Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade
C3  - Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia
T1  - In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density
SP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6249
ER  - 

@conference{
author = "Pajić, Tanja and Kozakijević, Suzana and Krmpot, Aleksandar J. and Živić, Miroslav and Todorović, Nataša and Rabasović, Mihailo D.",
year = "2023",
abstract = "Mitochondrial function, and consequently cellular metabolic status and fitness of a cell, is tightly linked
to the dynamic changes of mitochondrial morphology, including mitochondrial fusion, fission and
mitophagy [1]. Lipid droplets (LDs) can be in close contact with mitochondria, and accumulate
autophagy or mitophagy generated material during the reparatory processes [2]. The effect of increased
ambient temperature on mitochondrial morphology and LDs density in living cells of the filamentous
fungus Phycomyces blakesleeanus was investigated. For in vivo imaging of mitochondria and LDs
multiphoton microscopy was used. Multiphoton microscopy enables 3D imaging in high resolution and
reduced photodamage and photobleaching of the sample using IR ultrafast pulsed lasers. Mitochondria
were stained with the vital dye Rhodamine123 (Rh123) which enters these organelles based on their
membrane potential - mitochondria must be healthy/active to stain. A wavelength of 800 nm from Ti:Sa
laser (160 fs pulse duration, 76 MHz repetition rate) was used for two-photon excitation of Rh123. The
laser beam was focused by the Zeiss Plan Neofluar 40x1.3 objective lens and the signal was detected
through a bandpass interference filter MF530/43 (ThorLabs, USA). For LDs staining a Nile Red dye
was used and excited by Yb: KGW laser at 1040 nm [3]. Six morphological types of mitochondria were
observed in the hyphae of this fungus: intermediate type - normal, intermediate with small semicircular
tubules, tubular, elongated tubular, fragmented (small ellipsoid tubule) and fragmented with exclusively
spheroid-shaped mitochondria. Changes in mitochondrial morphology were induced by a small
temperature change. A 3°C increase in ambient temperature, from 22°C, had a dramatic effect on
mitochondrial morphology, inducing the appearance of a predominantly tubular mitochondrial
morphology. The total area percentage of mitochondria showed an increasing trend when grown at
25°C. Increasing the ambient temperature to 25°C induced a statistically significant increase in the
percentage of hyphal area occupied by LDs from 2.9 ± 1.6 to 4.7 ± 2.2 (mean value and SD in percentage
of hyphal area). The observed response to the small temperature increase points to the physiological
adaptation of hyphal metabolism.",
publisher = "Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade",
journal = "Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia",
title = "In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density",
pages = "84",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6249"
}

Pajić, T., Kozakijević, S., Krmpot, A. J., Živić, M., Todorović, N.,& Rabasović, M. D.. (2023). In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density. in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia
Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade., 84.
https://hdl.handle.net/21.15107/rcub_ibiss_6249

Pajić T, Kozakijević S, Krmpot AJ, Živić M, Todorović N, Rabasović MD. In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density. in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia. 2023;:84.
https://hdl.handle.net/21.15107/rcub_ibiss_6249 .

Pajić, Tanja, Kozakijević, Suzana, Krmpot, Aleksandar J., Živić, Miroslav, Todorović, Nataša, Rabasović, Mihailo D., "In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density" in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia (2023):84,
https://hdl.handle.net/21.15107/rcub_ibiss_6249 .

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  - Stevanović, Katarina S
AU  - Čepkenović, Bogdana
AU  - Križak, Strahinja
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav Ž
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6088
AB  - Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.
PB  - Springer Nature
T2  - Scientific Reports
T1  - ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore
IS  - 1
VL  - 13
DO  - 10.1038/s41598-023-39021-9
SP  - 11897
ER  - 

@article{
author = "Stevanović, Katarina S and Čepkenović, Bogdana and Križak, Strahinja and Pajić, Tanja and Todorović, Nataša and Živić, Miroslav Ž",
year = "2023",
abstract = "Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.",
publisher = "Springer Nature",
journal = "Scientific Reports",
title = "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore",
number = "1",
volume = "13",
doi = "10.1038/s41598-023-39021-9",
pages = "11897"
}

Stevanović, K. S., Čepkenović, B., Križak, S., Pajić, T., Todorović, N.,& Živić, M. Ž.. (2023). ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports
Springer Nature., 13(1), 11897.
https://doi.org/10.1038/s41598-023-39021-9

Stevanović KS, Čepkenović B, Križak S, Pajić T, Todorović N, Živić MŽ. ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports. 2023;13(1):11897.
doi:10.1038/s41598-023-39021-9 .

Stevanović, Katarina S, Čepkenović, Bogdana, Križak, Strahinja, Pajić, Tanja, Todorović, Nataša, Živić, Miroslav Ž, "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore" in Scientific Reports, 13, no. 1 (2023):11897,
https://doi.org/10.1038/s41598-023-39021-9 . .

TY  - JOUR
AU  - Stevanović, Katarina
AU  - Čepkenović, Bogdana
AU  - Križak, Strahinja
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5978
AB  - Studies of ion currents in filamentous fungi are a prerequisite for forming a complete
understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of
Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in
the native membrane, including the currents mediated by the channels not yet molecularly identified.
Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in
the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have
previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated
anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity
sequence, and time and voltage dependent profile of the current. Using the patch clamp method on
the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine
the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of
chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior
in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in
filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.
PB  - Basel: MDPI
T2  - Journal of Fungi
T1  - Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current
IS  - 6
VL  - 9
DO  - 10.3390/jof9060637
SP  - 637
ER  - 

@article{
author = "Stevanović, Katarina and Čepkenović, Bogdana and Križak, Strahinja and Živić, Miroslav and Todorović, Nataša",
year = "2023",
abstract = "Studies of ion currents in filamentous fungi are a prerequisite for forming a complete
understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of
Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in
the native membrane, including the currents mediated by the channels not yet molecularly identified.
Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in
the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have
previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated
anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity
sequence, and time and voltage dependent profile of the current. Using the patch clamp method on
the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine
the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of
chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior
in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in
filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.",
publisher = "Basel: MDPI",
journal = "Journal of Fungi",
title = "Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current",
number = "6",
volume = "9",
doi = "10.3390/jof9060637",
pages = "637"
}

Stevanović, K., Čepkenović, B., Križak, S., Živić, M.,& Todorović, N.. (2023). Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current. in Journal of Fungi
Basel: MDPI., 9(6), 637.
https://doi.org/10.3390/jof9060637

Stevanović K, Čepkenović B, Križak S, Živić M, Todorović N. Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current. in Journal of Fungi. 2023;9(6):637.
doi:10.3390/jof9060637 .

Stevanović, Katarina, Čepkenović, Bogdana, Križak, Strahinja, Živić, Miroslav, Todorović, Nataša, "Osmotically activated anion current of Phycomyces blakesleeanus-filamentous fungi counterpart to vertebrate volume regulated anion current" in Journal of Fungi, 9, no. 6 (2023):637,
https://doi.org/10.3390/jof9060637 . .

TY  - JOUR
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Nikolić, Stanko N
AU  - Rabasović, Mihailo D
AU  - Clayton, Andrew H A
AU  - Krmpot, Aleksandar J
PY  - 2022
UR  - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC9637149
UR  - https://www.nature.com/articles/s41598-022-23502-4
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5235
AB  - We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus . THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10 −3 to 23 × 10 −3 lipid droplets/µm 2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.
PB  - Berlin: Nature Portfolio
T2  - Scientific Reports
T1  - Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi
IS  - 1
VL  - 12
DO  - 10.1038/s41598-022-23502-4
SP  - 18760
ER  - 

@article{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Nikolić, Stanko N and Rabasović, Mihailo D and Clayton, Andrew H A and Krmpot, Aleksandar J",
year = "2022",
abstract = "We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus . THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10 −3 to 23 × 10 −3 lipid droplets/µm 2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.",
publisher = "Berlin: Nature Portfolio",
journal = "Scientific Reports",
title = "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi",
number = "1",
volume = "12",
doi = "10.1038/s41598-022-23502-4",
pages = "18760"
}

Pajić, T., Todorović, N., Živić, M., Nikolić, S. N., Rabasović, M. D., Clayton, A. H. A.,& Krmpot, A. J.. (2022). Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Scientific Reports
Berlin: Nature Portfolio., 12(1), 18760.
https://doi.org/10.1038/s41598-022-23502-4

Pajić T, Todorović N, Živić M, Nikolić SN, Rabasović MD, Clayton AHA, Krmpot AJ. Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Scientific Reports. 2022;12(1):18760.
doi:10.1038/s41598-022-23502-4 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Nikolić, Stanko N, Rabasović, Mihailo D, Clayton, Andrew H A, Krmpot, Aleksandar J, "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi" in Scientific Reports, 12, no. 1 (2022):18760,
https://doi.org/10.1038/s41598-022-23502-4 . .

TY  - JOUR
AU  - Živić, Ivana
AU  - Božanić, Milenka
AU  - Todorović, Nataša
AU  - Stojanović, Katarina
PY  - 2022
UR  - https://acta-zoologica-bulgarica.eu/june-2022/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5079
AB  - In the course of hydrobiological studies on the territory of Serbia in 2020, three species of the genus Branchiobdella (Annelida: Branchiobdellea) were recorded: B. parasita (Braun, 1805). B. pentadonta Whitman, 1882, and B. hexadonta Gruber. 1883. They were found on the stone crayfish Austropotamobius torrentiurn (Schrank. 1803) in the Magaka River (Delivoda locality, Southeast Serbia) and in the Rukjavica Stream (Eastern Serbia). This is the first record of the genus Branchiobdella and the three species on the territory' of Serbia.
PB  - Sofia: Institute of Biodiversity and Ecosystem Research – Bulgarian Academy of Sciences
T2  - Acta Zoologica Bulgarica
T1  - Branchiobdella Odier, 1823 ( Annelida : Branchiobdellea ), a New Genus for the Fauna of Serbia
IS  - 2
VL  - 74
SP  - 325
EP  - 327
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5079
ER  - 

@article{
author = "Živić, Ivana and Božanić, Milenka and Todorović, Nataša and Stojanović, Katarina",
year = "2022",
abstract = "In the course of hydrobiological studies on the territory of Serbia in 2020, three species of the genus Branchiobdella (Annelida: Branchiobdellea) were recorded: B. parasita (Braun, 1805). B. pentadonta Whitman, 1882, and B. hexadonta Gruber. 1883. They were found on the stone crayfish Austropotamobius torrentiurn (Schrank. 1803) in the Magaka River (Delivoda locality, Southeast Serbia) and in the Rukjavica Stream (Eastern Serbia). This is the first record of the genus Branchiobdella and the three species on the territory' of Serbia.",
publisher = "Sofia: Institute of Biodiversity and Ecosystem Research – Bulgarian Academy of Sciences",
journal = "Acta Zoologica Bulgarica",
title = "Branchiobdella Odier, 1823 ( Annelida : Branchiobdellea ), a New Genus for the Fauna of Serbia",
number = "2",
volume = "74",
pages = "325-327",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5079"
}

Živić, I., Božanić, M., Todorović, N.,& Stojanović, K.. (2022). Branchiobdella Odier, 1823 ( Annelida : Branchiobdellea ), a New Genus for the Fauna of Serbia. in Acta Zoologica Bulgarica
Sofia: Institute of Biodiversity and Ecosystem Research – Bulgarian Academy of Sciences., 74(2), 325-327.
https://hdl.handle.net/21.15107/rcub_ibiss_5079

Živić I, Božanić M, Todorović N, Stojanović K. Branchiobdella Odier, 1823 ( Annelida : Branchiobdellea ), a New Genus for the Fauna of Serbia. in Acta Zoologica Bulgarica. 2022;74(2):325-327.
https://hdl.handle.net/21.15107/rcub_ibiss_5079 .

Živić, Ivana, Božanić, Milenka, Todorović, Nataša, Stojanović, Katarina, "Branchiobdella Odier, 1823 ( Annelida : Branchiobdellea ), a New Genus for the Fauna of Serbia" in Acta Zoologica Bulgarica, 74, no. 2 (2022):325-327,
https://hdl.handle.net/21.15107/rcub_ibiss_5079 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Živić, Miroslav
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5742
AB  - Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz).
AB  - Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom
T1  - Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером
SP  - 24
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5742
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Živić, Miroslav and Krmpot, Aleksandar and Rabasović, Mihailo and Todorović, Nataša",
year = "2022",
abstract = "Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz)., Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom, Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером",
pages = "24",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5742"
}

Stevanović, K., Pajić, T., Živić, M., Krmpot, A., Rabasović, M.,& Todorović, N.. (2022). Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742

Stevanović K, Pajić T, Živić M, Krmpot A, Rabasović M, Todorović N. Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

Stevanović, Katarina, Pajić, Tanja, Živić, Miroslav, Krmpot, Aleksandar, Rabasović, Mihailo, Todorović, Nataša, "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):24,
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo
AU  - Krmpot, Aleksandar
PY  - 2022
UR  - http://www.photonicsworkshop.ipb.ac.rs/15/images/RFotonika2022KnjigaApstrakata-B5-CIP.pdf
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5735
AB  - Lipids in oleaginous filamentous fungi are considered to be a valuable alternative resource for various biotechnological applications. In vivo label-free imaging enables monitoring of fungi lipid droplets (LD) accumulation with the minimal unwanted effects on the metabolism. LDs are the main source of contrast [1] in the Third Harmonic Generation (THG) microscopy method [2] due to their optical properties (high refractive index). The THG phenomenon is utilized in nonlinear laser scanning microscopy that employs ultra-short laser pulses for imaging. We present in vivo and label-free THG imaging of the individual hyphae of Phycomyces blakesleeanus (Figure 1), an oleaginous filamentous fungus with very rapid growth rate. The THG signal was detected in the forward direction (transmission arm) by PMT through Hoya glass UV filter with peak transmission at 340 nm. The Yb KGW laser, wavelength 1040 nm, pulse duration 200 fs, and repetition rate 83 MHz, has been the source  of the infrared femtosecond pulses. The LDs from THG images were quantified by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting Particle Size Analysis (PSA). We used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and increase of cellular lipid reserve. The two analysis methods gave similar results. The applicability of the described procedure can be easily extended to other unicellular organisms for the in vivo quantification of LDs since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that the ICA is suitable for THG images, although it is primarily developed and have been mostly used for fluorescence signals so far. REFERENCES [1] D. Débarre, W. Supatto et al., Nat. Methods, 3(1), (2006), 47-53. [2] R. W. Boyd, Nonlinear Optics, Academic Press (2008).
PB  - Belgrade: Institute of Physics
C3  - Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia
T1  - Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi
SP  - 52
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5735
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Rabasović, Mihailo and Krmpot, Aleksandar",
year = "2022",
abstract = "Lipids in oleaginous filamentous fungi are considered to be a valuable alternative resource for various biotechnological applications. In vivo label-free imaging enables monitoring of fungi lipid droplets (LD) accumulation with the minimal unwanted effects on the metabolism. LDs are the main source of contrast [1] in the Third Harmonic Generation (THG) microscopy method [2] due to their optical properties (high refractive index). The THG phenomenon is utilized in nonlinear laser scanning microscopy that employs ultra-short laser pulses for imaging. We present in vivo and label-free THG imaging of the individual hyphae of Phycomyces blakesleeanus (Figure 1), an oleaginous filamentous fungus with very rapid growth rate. The THG signal was detected in the forward direction (transmission arm) by PMT through Hoya glass UV filter with peak transmission at 340 nm. The Yb KGW laser, wavelength 1040 nm, pulse duration 200 fs, and repetition rate 83 MHz, has been the source  of the infrared femtosecond pulses. The LDs from THG images were quantified by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting Particle Size Analysis (PSA). We used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and increase of cellular lipid reserve. The two analysis methods gave similar results. The applicability of the described procedure can be easily extended to other unicellular organisms for the in vivo quantification of LDs since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that the ICA is suitable for THG images, although it is primarily developed and have been mostly used for fluorescence signals so far. REFERENCES [1] D. Débarre, W. Supatto et al., Nat. Methods, 3(1), (2006), 47-53. [2] R. W. Boyd, Nonlinear Optics, Academic Press (2008).",
publisher = "Belgrade: Institute of Physics",
journal = "Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia",
title = "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi",
pages = "52",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5735"
}

Pajić, T., Todorović, N., Živić, M., Rabasović, M.,& Krmpot, A.. (2022). Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia
Belgrade: Institute of Physics., 52.
https://hdl.handle.net/21.15107/rcub_ibiss_5735

Pajić T, Todorović N, Živić M, Rabasović M, Krmpot A. Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia. 2022;:52.
https://hdl.handle.net/21.15107/rcub_ibiss_5735 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Rabasović, Mihailo, Krmpot, Aleksandar, "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi" in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia (2022):52,
https://hdl.handle.net/21.15107/rcub_ibiss_5735 .

TY  - CONF
AU  - Žižić, Milan
AU  - Stanić, Marina
AU  - Aquilanti, Giuliana
AU  - Cvetić Antić, Tijana
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Zakrzewska, Joanna
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5733
AB  - In this study, mycelium of fungus Phycomyces blakesleeanus was exposed to soluble toxic form of selenium, selenite (Se+4), with the aim of determining the flow and products of its biotransformation. Selenite is reduced to Se0 in the form selenium nanoparticles (SeNPs) and Se-2 in the form of methylated volatile selenides. Low concentrations of Se+4 in the mycelium form RSSeSR type compounds, which could be a step in the process of SeNPs formation, or incorporation of Se into metabolites such as Se-amino acids.
PB  - Belgrade: Society of Physical Chemists of Serbia
C3  - Proceedings: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry: Physical Chemistry 2022, Vol. 1; 2022 Sep 26-30; Belgrade, Serbia
T1  - Selenite metabolism in the mycelium of the fungus Phycomyces blakesleeanus
SP  - 93
EP  - 96
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5733
ER  - 

@conference{
author = "Žižić, Milan and Stanić, Marina and Aquilanti, Giuliana and Cvetić Antić, Tijana and Todorović, Nataša and Živić, Miroslav and Zakrzewska, Joanna",
year = "2022",
abstract = "In this study, mycelium of fungus Phycomyces blakesleeanus was exposed to soluble toxic form of selenium, selenite (Se+4), with the aim of determining the flow and products of its biotransformation. Selenite is reduced to Se0 in the form selenium nanoparticles (SeNPs) and Se-2 in the form of methylated volatile selenides. Low concentrations of Se+4 in the mycelium form RSSeSR type compounds, which could be a step in the process of SeNPs formation, or incorporation of Se into metabolites such as Se-amino acids.",
publisher = "Belgrade: Society of Physical Chemists of Serbia",
journal = "Proceedings: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry: Physical Chemistry 2022, Vol. 1; 2022 Sep 26-30; Belgrade, Serbia",
title = "Selenite metabolism in the mycelium of the fungus Phycomyces blakesleeanus",
pages = "93-96",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5733"
}

Žižić, M., Stanić, M., Aquilanti, G., Cvetić Antić, T., Todorović, N., Živić, M.,& Zakrzewska, J.. (2022). Selenite metabolism in the mycelium of the fungus Phycomyces blakesleeanus. in Proceedings: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry: Physical Chemistry 2022, Vol. 1; 2022 Sep 26-30; Belgrade, Serbia
Belgrade: Society of Physical Chemists of Serbia., 93-96.
https://hdl.handle.net/21.15107/rcub_ibiss_5733

Žižić M, Stanić M, Aquilanti G, Cvetić Antić T, Todorović N, Živić M, Zakrzewska J. Selenite metabolism in the mycelium of the fungus Phycomyces blakesleeanus. in Proceedings: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry: Physical Chemistry 2022, Vol. 1; 2022 Sep 26-30; Belgrade, Serbia. 2022;:93-96.
https://hdl.handle.net/21.15107/rcub_ibiss_5733 .

Žižić, Milan, Stanić, Marina, Aquilanti, Giuliana, Cvetić Antić, Tijana, Todorović, Nataša, Živić, Miroslav, Zakrzewska, Joanna, "Selenite metabolism in the mycelium of the fungus Phycomyces blakesleeanus" in Proceedings: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry: Physical Chemistry 2022, Vol. 1; 2022 Sep 26-30; Belgrade, Serbia (2022):93-96,
https://hdl.handle.net/21.15107/rcub_ibiss_5733 .

TY  - CONF
AU  - Milićević, Katarina
AU  - Lazarević, Milica
AU  - Momčilović, Miljana
AU  - Todorović, Nataša
AU  - Petković, Branka
AU  - Stojadinović, Gordana
AU  - Nikolić, Ljiljana
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5504
AB  - Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS)
characterized by infiltration of lymphocytes that leads to myelin damage and neurodegeneration.
The complex interaction between CNS-infiltrating immune cells (CNS-IIC) and astrocytes is an
important contributor to the disease progression. Here, we investigate how naïve astrocytes respond
to autoreactive immune cells present in the CNS at different stages of the disease. For this purpose,
CNS-IICs were isolated from the spinal cords of rats with experimental autoimmune
encephalomyelitis at onset, late-onset and the peak of the disease. Naïve astrocytes, isolated from
the spinal cords of wild-type rat pups, responded to brief bath application of CNS-IIC by robust
elevation of intracellular Ca2+ independently of the disease stage. Our data suggest that direct
contact between astrocytes and CNS-IICs induces Ca2+ changes in astrocytes and points to the new
aspect of cell-cell interactions in the propagation of neuroinflammatory response in CNS
autoimmunity.
PB  - Belgrade: Society of Physical Chemists of Serbia
C3  - Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia
T1  - Naïve astrocytes react to CNS-infiltrated immune cells
SP  - 267
EP  - 270
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5504
ER  - 

@conference{
editor = "Čupić, Željko, Anić, Slobodan",
author = "Milićević, Katarina and Lazarević, Milica and Momčilović, Miljana and Todorović, Nataša and Petković, Branka and Stojadinović, Gordana and Nikolić, Ljiljana",
year = "2022",
abstract = "Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS)
characterized by infiltration of lymphocytes that leads to myelin damage and neurodegeneration.
The complex interaction between CNS-infiltrating immune cells (CNS-IIC) and astrocytes is an
important contributor to the disease progression. Here, we investigate how naïve astrocytes respond
to autoreactive immune cells present in the CNS at different stages of the disease. For this purpose,
CNS-IICs were isolated from the spinal cords of rats with experimental autoimmune
encephalomyelitis at onset, late-onset and the peak of the disease. Naïve astrocytes, isolated from
the spinal cords of wild-type rat pups, responded to brief bath application of CNS-IIC by robust
elevation of intracellular Ca2+ independently of the disease stage. Our data suggest that direct
contact between astrocytes and CNS-IICs induces Ca2+ changes in astrocytes and points to the new
aspect of cell-cell interactions in the propagation of neuroinflammatory response in CNS
autoimmunity.",
publisher = "Belgrade: Society of Physical Chemists of Serbia",
journal = "Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia",
title = "Naïve astrocytes react to CNS-infiltrated immune cells",
pages = "267-270",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5504"
}

Čupić, Ž., Anić, S., Milićević, K., Lazarević, M., Momčilović, M., Todorović, N., Petković, B., Stojadinović, G.,& Nikolić, L.. (2022). Naïve astrocytes react to CNS-infiltrated immune cells. in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia
Belgrade: Society of Physical Chemists of Serbia., 267-270.
https://hdl.handle.net/21.15107/rcub_ibiss_5504

Čupić Ž, Anić S, Milićević K, Lazarević M, Momčilović M, Todorović N, Petković B, Stojadinović G, Nikolić L. Naïve astrocytes react to CNS-infiltrated immune cells. in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia. 2022;:267-270.
https://hdl.handle.net/21.15107/rcub_ibiss_5504 .

Čupić, Željko, Anić, Slobodan, Milićević, Katarina, Lazarević, Milica, Momčilović, Miljana, Todorović, Nataša, Petković, Branka, Stojadinović, Gordana, Nikolić, Ljiljana, "Naïve astrocytes react to CNS-infiltrated immune cells" in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia (2022):267-270,
https://hdl.handle.net/21.15107/rcub_ibiss_5504 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Kozakijević, Suzana
AU  - Lukičić, Jovana
AU  - Živić, Miroslav
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5744
AB  - Biološka aktivnost selena zavisi od njegove doze i hemijskog oblika. U mikrotragovima povećava antioksidativni kapacitet, dok je u višim koncentracijama toksičan zbog stvaranja reaktivnih vrsta kiseonika i potencijalnog oštećenja mitohondrijalne membrane. Končaste gljive imaju sposobnost koncentrisanja selena u micelijumu i predstavljaju jedan od glavnih puteva za njegov ulazak u ekosisteme. U ovom radu je ispitivan uticaj hroničnog i akutnog dejstva natrijum selenita (uobičajen oblik selena u ishrani) na morfologiju i zastupljenost mitohondrija u živim ćelijama končaste gljive Phycomyces blakesleeanus tokom eksponencijalne faze rasta (EFR). Za in vivo oslikavanje morfologije mitohondrija i njihove dinamike u odgovoru na različite koncentracije selenita korišćena je multifotonska mikroskopija koja omogućava trodimenzionalno oslikavanje u visokoj rezoluciji i smanjeno fotooštećenje i fotoizbeljivanje uzorka zahvaljujući upotrebi infracrvenih ultrabrzih impulsnih lasera. Hronični tretmani selenitom su doveli do uočljivog trenda smanjenja zastupljenosti mitohondrija sa povećanjem koncentracije selenita. Izloženost hifa selenitu, tokom akutnih i hroničnih tretmana, rezultiralo je povećanjem zastupljenosti tubularne morfologije mitohondrija, koja je u akutnom tretmanu većim koncentracijama selenita (0,75 i 1 mM) bila povećana za 50% u odnosu na kontrolu. Kod mlađih hifa EFR izloženih hroničnom delovanju selenita (0,5 do 1 mM) tubularne mitohondrije su bile višestruko duže od onih u starijoj populaciji.
AB  - Биолошка активност селена зависи од његове дозе и хемијског облика. У
микротраговима повећава антиоксидативни капацитет, док је у вишим
концентрацијама токсичан због стварања реактивних врста кисеоника и
потенцијалног оштећења митохондријалне мембране. Кончасте гљиве имају
способност концентрисања селена у мицелијуму и представљају један од главних
путева за његов улазак у екосистеме. У овом раду је испитиван утицај хроничног и
акутног дејства натријум селенита (уобичајен облик селена у исхрани) на
морфологију и заступљеност митохондрија у живим ћелијама кончасте гљиве
Phycomyces blakesleeanus током експоненцијалне фазе раста (ЕФР). За in vivo
осликавање морфологије митохондрија и њихове динамике у одговору на
различите концентрације селенита коришћена је мултифотонска микроскопија која
омогућава тродимензионално осликавање у високој резолуцији и смањено
фотооштећење и фотоизбељивање узорка захваљујући употреби инфрацрвених
ултрабрзих импулсних ласера. Хронични третмани селенитом су довели до
уочљивог тренда смањења заступљености митохондрија са повећањем
концентрације селенита. Изложеност хифа селениту, током акутних и хроничних
третмана, резултирало је повећањем заступљености тубуларне морфологије
митохондрија, која је у акутном третману већим концентрацијама селенита (0,75 и
1 mМ) била повећана за 50% у односу на контролу. Код млађих хифа ЕФР
изложених хроничном деловању селенита (0,5 до 1 mМ) тубуларне митохондрије
су биле вишеструко дуже од оних у старијој популацији.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija
T1  - In vivo осликавање појединачних хифа Phycomyces blakesleeanus методом мултифотонске микроскопије: утицај селенита на морфологију и функцију митохондрија
SP  - 28
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5744
ER  - 

@conference{
author = "Pajić, Tanja and Kozakijević, Suzana and Lukičić, Jovana and Živić, Miroslav and Krmpot, Aleksandar and Rabasović, Mihailo and Todorović, Nataša",
year = "2022",
abstract = "Biološka aktivnost selena zavisi od njegove doze i hemijskog oblika. U mikrotragovima povećava antioksidativni kapacitet, dok je u višim koncentracijama toksičan zbog stvaranja reaktivnih vrsta kiseonika i potencijalnog oštećenja mitohondrijalne membrane. Končaste gljive imaju sposobnost koncentrisanja selena u micelijumu i predstavljaju jedan od glavnih puteva za njegov ulazak u ekosisteme. U ovom radu je ispitivan uticaj hroničnog i akutnog dejstva natrijum selenita (uobičajen oblik selena u ishrani) na morfologiju i zastupljenost mitohondrija u živim ćelijama končaste gljive Phycomyces blakesleeanus tokom eksponencijalne faze rasta (EFR). Za in vivo oslikavanje morfologije mitohondrija i njihove dinamike u odgovoru na različite koncentracije selenita korišćena je multifotonska mikroskopija koja omogućava trodimenzionalno oslikavanje u visokoj rezoluciji i smanjeno fotooštećenje i fotoizbeljivanje uzorka zahvaljujući upotrebi infracrvenih ultrabrzih impulsnih lasera. Hronični tretmani selenitom su doveli do uočljivog trenda smanjenja zastupljenosti mitohondrija sa povećanjem koncentracije selenita. Izloženost hifa selenitu, tokom akutnih i hroničnih tretmana, rezultiralo je povećanjem zastupljenosti tubularne morfologije mitohondrija, koja je u akutnom tretmanu većim koncentracijama selenita (0,75 i 1 mM) bila povećana za 50% u odnosu na kontrolu. Kod mlađih hifa EFR izloženih hroničnom delovanju selenita (0,5 do 1 mM) tubularne mitohondrije su bile višestruko duže od onih u starijoj populaciji., Биолошка активност селена зависи од његове дозе и хемијског облика. У
микротраговима повећава антиоксидативни капацитет, док је у вишим
концентрацијама токсичан због стварања реактивних врста кисеоника и
потенцијалног оштећења митохондријалне мембране. Кончасте гљиве имају
способност концентрисања селена у мицелијуму и представљају један од главних
путева за његов улазак у екосистеме. У овом раду је испитиван утицај хроничног и
акутног дејства натријум селенита (уобичајен облик селена у исхрани) на
морфологију и заступљеност митохондрија у живим ћелијама кончасте гљиве
Phycomyces blakesleeanus током експоненцијалне фазе раста (ЕФР). За in vivo
осликавање морфологије митохондрија и њихове динамике у одговору на
различите концентрације селенита коришћена је мултифотонска микроскопија која
омогућава тродимензионално осликавање у високој резолуцији и смањено
фотооштећење и фотоизбељивање узорка захваљујући употреби инфрацрвених
ултрабрзих импулсних ласера. Хронични третмани селенитом су довели до
уочљивог тренда смањења заступљености митохондрија са повећањем
концентрације селенита. Изложеност хифа селениту, током акутних и хроничних
третмана, резултирало је повећањем заступљености тубуларне морфологије
митохондрија, која је у акутном третману већим концентрацијама селенита (0,75 и
1 mМ) била повећана за 50% у односу на контролу. Код млађих хифа ЕФР
изложених хроничном деловању селенита (0,5 до 1 mМ) тубуларне митохондрије
су биле вишеструко дуже од оних у старијој популацији.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija, In vivo осликавање појединачних хифа Phycomyces blakesleeanus методом мултифотонске микроскопије: утицај селенита на морфологију и функцију митохондрија",
pages = "28",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5744"
}

Pajić, T., Kozakijević, S., Lukičić, J., Živić, M., Krmpot, A., Rabasović, M.,& Todorović, N.. (2022). In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 28.
https://hdl.handle.net/21.15107/rcub_ibiss_5744

Pajić T, Kozakijević S, Lukičić J, Živić M, Krmpot A, Rabasović M, Todorović N. In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:28.
https://hdl.handle.net/21.15107/rcub_ibiss_5744 .

Pajić, Tanja, Kozakijević, Suzana, Lukičić, Jovana, Živić, Miroslav, Krmpot, Aleksandar, Rabasović, Mihailo, Todorović, Nataša, "In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):28,
https://hdl.handle.net/21.15107/rcub_ibiss_5744 .

TY  - CONF
AU  - Bošnjaković, Tamara
AU  - Todorović, Nataša
AU  - Milošević, Milena
AU  - Živić, Miroslav
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5743
AB  - Prihvaćeni model lučenja insulina, po stimulaciji glukozom β ćelija pankreasa, čine: (1) transport glukoze u ćeliju; (2) KATP kanali čija blokada ATP-om dovodi do depolarizacije membrane; (3) voltažno-zavisni kalcijumski kanali koji se aktiviraju depolarizacijom, dozvoljavajući ulazak Ca2+; (4) egzocitoza insulinskih granula pokrenuta Ca2+ jonima.1 Pošto za dostizanje praga aktivacije nije dovoljno samo zatvaranje KATP kanala, jasno je da je uključena i aktivnost drugih jonskih kanala. Ima podataka da je ovaj proces posredovan kanalom VRAC (Volume-Regulated Anion Channel) koji provodi jone hlora i osmolite i aktivira se u hipotoničnoj sredini.2 Mi smo koristili metodu nametnute voltaže na deliću membrane uz oslikavanje signala Ca2+ indikatora, kako bi ispitali prisustvo i ulogu VRAC u električnoj aktivnosti RIN – 5f ćelija, u cilju utvrđivanja potencijalnih ograničenja i optimizacije eksperimentalnog pristupa izučavanja strujnog odgovora β ćelija. U prisustvu cezijuma (Cs+ pip), kojim se blokiraju K kanali, snimljene su familije struja u izotoničnim i hipotoničnim uslovima, kao i spontana i depolarizacijom indukovana aktivnost (akcioni potencijali i depolarizacija u odgovoru na stimulaciju glukozom). Testiran je efekat jedinog postojećeg farmakološkog alata za blokadu VRAC, DCPIB na membranske struje i na Ca2+ odgovor ćelija. Rezultati ukazuju da: (1) u RIN – 5f ćelijama nema inaktivirajućeg oblika VRAC i da hipotonični uslovi, u odsustvu glukoze, ne dovode do aktivacije struja; (2) u izotoničnim uslovima, DCPIB pored očekivanog delimičnog bloka struja ima i stimulativni efekat na druge izlazne struje; (3) preliminarna oslikavanja Ca2+ signala ukazuju da DCPIB inhibira a njegovo uklanjanje povećava aktivnost u prisustvu 0-15 mM glukoze.
AB  - Прихваћени модел лучења инсулина, по стимулацији глукозом β ћелија панкреаса,
чине: (1) транспорт глукозе у ћелију; (2) KATP канали чија блокада ATP-ом доводи
до деполаризације мембране; (3) волтажно-зависни калцијумски канали који се
активирају деполаризацијом, дозвољавајући улазак Ca2+; (4) егзоцитоза
инсулинских гранула покренута Ca2+ јонима.1 Пошто за достизање прага
активације није довољно само затварање KATP канала, јасно је да је укључена и
активност других јонских канала. Има података да је овај процес посредован
каналом VRAC (Volume-Regulated Anion Channel) који проводи јоне хлора и
осмолите и активира се у хипотоничној средини.2 Ми смо користили методу
наметнуте волтаже на делићу мембране уз осликавање сигнала Ca2+ индикатора,
како би испитали присуство и улогу VRAC у електричној активности RIN – 5f
ћелија, у циљу утврђивања потенцијалних ограничења и оптимизације
експерименталног приступа изучавања струјног одговора β ћелија. У присуству
цезијума (Cs+pip), којим се блокирају K канали, снимљене су фамилије струја у
изотоничним и хипотоничним условима, као и спонтана и деполаризацијом
индукована активност (акциони потенцијали и деполаризација у одговору на
стимулацију глукозом). Тестиран је ефекат јединог постојећег фармаколошког
алата за блокаду VRAC, DCPIB на мембранске струје и на Ca2+ одговор ћелија.
Резултати указују да: (1) у RIN – 5f ћелијама нема инактивирајућег облика VRAC и
да хипотонични услови, у одсуству глукозе, не доводе до активације струја; (2) у
изотоничним условима, DCPIB поред очекиваног делимичног блока струја има и
стимулативни ефекат на друге излазне струје; (3) прелиминарна осликавања Ca2+
сигнала указују да DCPIB инхибира а његово уклањање повећава активност у
присуству 0-15 mM глукозе.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Elektrofiziološka karakterizacija β ćelija pankreasa na ćelijskoj liniji RIN – 5f
T1  - Електрофизиолошка карактеризација β ћелија панкреаса на ћелијској линији RIN – 5f
SP  - 27
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5743
ER  - 

@conference{
author = "Bošnjaković, Tamara and Todorović, Nataša and Milošević, Milena and Živić, Miroslav",
year = "2022",
abstract = "Prihvaćeni model lučenja insulina, po stimulaciji glukozom β ćelija pankreasa, čine: (1) transport glukoze u ćeliju; (2) KATP kanali čija blokada ATP-om dovodi do depolarizacije membrane; (3) voltažno-zavisni kalcijumski kanali koji se aktiviraju depolarizacijom, dozvoljavajući ulazak Ca2+; (4) egzocitoza insulinskih granula pokrenuta Ca2+ jonima.1 Pošto za dostizanje praga aktivacije nije dovoljno samo zatvaranje KATP kanala, jasno je da je uključena i aktivnost drugih jonskih kanala. Ima podataka da je ovaj proces posredovan kanalom VRAC (Volume-Regulated Anion Channel) koji provodi jone hlora i osmolite i aktivira se u hipotoničnoj sredini.2 Mi smo koristili metodu nametnute voltaže na deliću membrane uz oslikavanje signala Ca2+ indikatora, kako bi ispitali prisustvo i ulogu VRAC u električnoj aktivnosti RIN – 5f ćelija, u cilju utvrđivanja potencijalnih ograničenja i optimizacije eksperimentalnog pristupa izučavanja strujnog odgovora β ćelija. U prisustvu cezijuma (Cs+ pip), kojim se blokiraju K kanali, snimljene su familije struja u izotoničnim i hipotoničnim uslovima, kao i spontana i depolarizacijom indukovana aktivnost (akcioni potencijali i depolarizacija u odgovoru na stimulaciju glukozom). Testiran je efekat jedinog postojećeg farmakološkog alata za blokadu VRAC, DCPIB na membranske struje i na Ca2+ odgovor ćelija. Rezultati ukazuju da: (1) u RIN – 5f ćelijama nema inaktivirajućeg oblika VRAC i da hipotonični uslovi, u odsustvu glukoze, ne dovode do aktivacije struja; (2) u izotoničnim uslovima, DCPIB pored očekivanog delimičnog bloka struja ima i stimulativni efekat na druge izlazne struje; (3) preliminarna oslikavanja Ca2+ signala ukazuju da DCPIB inhibira a njegovo uklanjanje povećava aktivnost u prisustvu 0-15 mM glukoze., Прихваћени модел лучења инсулина, по стимулацији глукозом β ћелија панкреаса,
чине: (1) транспорт глукозе у ћелију; (2) KATP канали чија блокада ATP-ом доводи
до деполаризације мембране; (3) волтажно-зависни калцијумски канали који се
активирају деполаризацијом, дозвољавајући улазак Ca2+; (4) егзоцитоза
инсулинских гранула покренута Ca2+ јонима.1 Пошто за достизање прага
активације није довољно само затварање KATP канала, јасно је да је укључена и
активност других јонских канала. Има података да је овај процес посредован
каналом VRAC (Volume-Regulated Anion Channel) који проводи јоне хлора и
осмолите и активира се у хипотоничној средини.2 Ми смо користили методу
наметнуте волтаже на делићу мембране уз осликавање сигнала Ca2+ индикатора,
како би испитали присуство и улогу VRAC у електричној активности RIN – 5f
ћелија, у циљу утврђивања потенцијалних ограничења и оптимизације
експерименталног приступа изучавања струјног одговора β ћелија. У присуству
цезијума (Cs+pip), којим се блокирају K канали, снимљене су фамилије струја у
изотоничним и хипотоничним условима, као и спонтана и деполаризацијом
индукована активност (акциони потенцијали и деполаризација у одговору на
стимулацију глукозом). Тестиран је ефекат јединог постојећег фармаколошког
алата за блокаду VRAC, DCPIB на мембранске струје и на Ca2+ одговор ћелија.
Резултати указују да: (1) у RIN – 5f ћелијама нема инактивирајућег облика VRAC и
да хипотонични услови, у одсуству глукозе, не доводе до активације струја; (2) у
изотоничним условима, DCPIB поред очекиваног делимичног блока струја има и
стимулативни ефекат на друге излазне струје; (3) прелиминарна осликавања Ca2+
сигнала указују да DCPIB инхибира а његово уклањање повећава активност у
присуству 0-15 mM глукозе.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Elektrofiziološka karakterizacija β ćelija pankreasa na ćelijskoj liniji RIN – 5f, Електрофизиолошка карактеризација β ћелија панкреаса на ћелијској линији RIN – 5f",
pages = "27",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5743"
}

Bošnjaković, T., Todorović, N., Milošević, M.,& Živić, M.. (2022). Elektrofiziološka karakterizacija β ćelija pankreasa na ćelijskoj liniji RIN – 5f. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 27.
https://hdl.handle.net/21.15107/rcub_ibiss_5743

Bošnjaković T, Todorović N, Milošević M, Živić M. Elektrofiziološka karakterizacija β ćelija pankreasa na ćelijskoj liniji RIN – 5f. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:27.
https://hdl.handle.net/21.15107/rcub_ibiss_5743 .

Bošnjaković, Tamara, Todorović, Nataša, Milošević, Milena, Živić, Miroslav, "Elektrofiziološka karakterizacija β ćelija pankreasa na ćelijskoj liniji RIN – 5f" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):27,
https://hdl.handle.net/21.15107/rcub_ibiss_5743 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5741
AB  - Izlazno-rektifikujuća inaktivirajuća struja (IRIS) je dominantna anjonska struja u uslovima osmotskog stresa na membrani poreklom iz sporangiofora gljive Phycomyces blakesleeanus.1 Jedna od njenih upečatljivih karakteristika je obrazac inaktivacije na depolarišućim potencijalima.1 Sa gubitkom adenozin trifosfata (ATF) sa unutarćelijske strane tokom dijalize pipetnim rastvorom, inaktivacija se vremenom ubrzava i dovodi do nepovratnog pada amplitude ukupne struje, dok se sa ATF-om prisutnim u pipetnom rastvoru amplituda struje i konstanta inaktivacije τ održavaju u vremenu.2 Naši najskoriji rezultati ukazuju da je, u prisustvu unutarćelijskog ATF-a oporavak od inaktivacije izazvane depolarizacijom voltažno- i vremenski-zavisan: (1) izlaganje hiperpolarizaciji je neophodno za dostizanje oporavka ≥ 90%; (2) procenat oporavka pri minimalnom vremenu odmora raste sa dubinom hiperpolarizacije; (3) brzina eksponencijalnog pada amplitude pri skraćenju vremena oporavka opada sa dubinom hiperpolarizacije. U uslovima smanjene koncentracije ATF-a koja je postignuta preinkubacijom natrijum-azidom, već u prvom minutu po ulasku u konfiguraciju „cela ćelija“ uočena je ubrzana inaktivacija.2 Dalja analiza voltažne-zavisnosti τ inaktivacije IRIS u različitim uslovima jasno pokazuje pomeraj ka manjim vrednostima τ sa opadanjem očekivane koncentracije ATF-a dok je voltažna zavisnost neznatno izmenjena. IRIS je identifikovana i u istrgnutoj konfiguraciji („spolja spolja“), pri čemu je obrazac oporavka od inaktivacije poslužio kao dodatna potvrda da su registrovana pojedinačna otvaranja upravo kanali u osnovi IRIS.
AB  - Излазно-ректификујућа инактивирајућа струја (ИРИС) је доминантна анјонска
струја у условима осмотског стреса на мембрани пореклом из спорангиофора
гљиве Phycomyces blakesleeanus.1 Једна од њених упечатљивих карактеристика је
образац инактивације на деполаришућим потенцијалима.1 Са губитком аденозин
трифосфата (АТФ) са унутарћелијске стране током дијализе пипетним раствором,
инактивација се временом убрзава и доводи до неповратног пада амплитуде укупне
струје, док се са АТФ-ом присутним у пипетном раствору амплитуда струје и
константа инактивације τ одржавају у времену.2 Наши најскорији резултати указују
да је, у присуству унутарћелијског АТФ-а опоравак од инактивације изазване
деполаризацијом волтажно- и временски-зависан: (1) излагање хиперполаризацији
је неопходно за достизање опоравка ≥ 90%; (2) проценат опоравка при минималном
времену одмора расте са дубином хиперполаризације; (3) брзина експоненцијалног
пада амплитуде при скраћењу времена опоравка опада са дубином
хиперполаризације. У условима смањене концентрације АТФ-а која је постигнута
преинкубацијом натријум-азидом, већ у првом минуту по уласку у конфигурацију
„цела ћелија“ уочена је убрзана инактивација.2 Даља анализа волтажне-зависности
τ инактивације ИРИС у различитим условима јасно показује померај ка мањим
вредностима τ са опадањем очекиване концентрације АТФ-а док је волтажна
зависност незнатно измењена. ИРИС је идентификована и у истргнутој
конфигурацији („споља споља“), при чему је образац опоравка од инактивације
послужио као додатна потврда да су регистрована појединачна отварања управо
канали у основи ИРИС.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus
T1  - Биофизички профил процеса инактивације излазно-ректификујуће инактивирајуће струје гљиве Phycomyces blakesleeanus
SP  - 23
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5741
ER  - 

@conference{
author = "Stevanović, Katarina and Živić, Miroslav and Todorović, Nataša",
year = "2022",
abstract = "Izlazno-rektifikujuća inaktivirajuća struja (IRIS) je dominantna anjonska struja u uslovima osmotskog stresa na membrani poreklom iz sporangiofora gljive Phycomyces blakesleeanus.1 Jedna od njenih upečatljivih karakteristika je obrazac inaktivacije na depolarišućim potencijalima.1 Sa gubitkom adenozin trifosfata (ATF) sa unutarćelijske strane tokom dijalize pipetnim rastvorom, inaktivacija se vremenom ubrzava i dovodi do nepovratnog pada amplitude ukupne struje, dok se sa ATF-om prisutnim u pipetnom rastvoru amplituda struje i konstanta inaktivacije τ održavaju u vremenu.2 Naši najskoriji rezultati ukazuju da je, u prisustvu unutarćelijskog ATF-a oporavak od inaktivacije izazvane depolarizacijom voltažno- i vremenski-zavisan: (1) izlaganje hiperpolarizaciji je neophodno za dostizanje oporavka ≥ 90%; (2) procenat oporavka pri minimalnom vremenu odmora raste sa dubinom hiperpolarizacije; (3) brzina eksponencijalnog pada amplitude pri skraćenju vremena oporavka opada sa dubinom hiperpolarizacije. U uslovima smanjene koncentracije ATF-a koja je postignuta preinkubacijom natrijum-azidom, već u prvom minutu po ulasku u konfiguraciju „cela ćelija“ uočena je ubrzana inaktivacija.2 Dalja analiza voltažne-zavisnosti τ inaktivacije IRIS u različitim uslovima jasno pokazuje pomeraj ka manjim vrednostima τ sa opadanjem očekivane koncentracije ATF-a dok je voltažna zavisnost neznatno izmenjena. IRIS je identifikovana i u istrgnutoj konfiguraciji („spolja spolja“), pri čemu je obrazac oporavka od inaktivacije poslužio kao dodatna potvrda da su registrovana pojedinačna otvaranja upravo kanali u osnovi IRIS., Излазно-ректификујућа инактивирајућа струја (ИРИС) је доминантна анјонска
струја у условима осмотског стреса на мембрани пореклом из спорангиофора
гљиве Phycomyces blakesleeanus.1 Једна од њених упечатљивих карактеристика је
образац инактивације на деполаришућим потенцијалима.1 Са губитком аденозин
трифосфата (АТФ) са унутарћелијске стране током дијализе пипетним раствором,
инактивација се временом убрзава и доводи до неповратног пада амплитуде укупне
струје, док се са АТФ-ом присутним у пипетном раствору амплитуда струје и
константа инактивације τ одржавају у времену.2 Наши најскорији резултати указују
да је, у присуству унутарћелијског АТФ-а опоравак од инактивације изазване
деполаризацијом волтажно- и временски-зависан: (1) излагање хиперполаризацији
је неопходно за достизање опоравка ≥ 90%; (2) проценат опоравка при минималном
времену одмора расте са дубином хиперполаризације; (3) брзина експоненцијалног
пада амплитуде при скраћењу времена опоравка опада са дубином
хиперполаризације. У условима смањене концентрације АТФ-а која је постигнута
преинкубацијом натријум-азидом, већ у првом минуту по уласку у конфигурацију
„цела ћелија“ уочена је убрзана инактивација.2 Даља анализа волтажне-зависности
τ инактивације ИРИС у различитим условима јасно показује померај ка мањим
вредностима τ са опадањем очекиване концентрације АТФ-а док је волтажна
зависност незнатно измењена. ИРИС је идентификована и у истргнутој
конфигурацији („споља споља“), при чему је образац опоравка од инактивације
послужио као додатна потврда да су регистрована појединачна отварања управо
канали у основи ИРИС.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus, Биофизички профил процеса инактивације излазно-ректификујуће инактивирајуће струје гљиве Phycomyces blakesleeanus",
pages = "23",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5741"
}

Stevanović, K., Živić, M.,& Todorović, N.. (2022). Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 23.
https://hdl.handle.net/21.15107/rcub_ibiss_5741

Stevanović K, Živić M, Todorović N. Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:23.
https://hdl.handle.net/21.15107/rcub_ibiss_5741 .

Stevanović, Katarina, Živić, Miroslav, Todorović, Nataša, "Biofizički profil procesa inaktivacije izlazno-rektifikujuće inaktivirajuće struje gljive Phycomyces blakesleeanus" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):23,
https://hdl.handle.net/21.15107/rcub_ibiss_5741 .

TY  - CONF
AU  - Nikolić, Ljiljana
AU  - Vidović, Marija
AU  - Todorović, Nataša
AU  - Petković, Branka
AU  - Stojadinović, Gordana
AU  - Martać, Ljiljana
AU  - Bogdanović Pristov, Jelena
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5508
AB  - Investigating membrane properties of plants is a challenging task, considering that success of
experiments is highly dependent on the possibility to isolate metabolically active protoplasts that
can withstand membrane current recordings. The aim of the present work is to obtain viable
protoplasts derived from root cells of Pisum sativum that can be used for the whole-cell patchclamp. We designed the procedure of the pea protoplasts isolation that delivers stable protoplasts
with preserved membrane integrity suitable for electrophysiological experiments. We applied a
custom approach for patch-clamping protoplasts using a microscope with a movable microscope
stage. We recorded prominent inward and prominent outward types of membrane current profiles of
protoplasts. Obtained data indicate that optimized isolation protocol and custom system for patchclamping, can be applied to study membrane properties of root protoplasts.
PB  - Belgrade: Society of Physical Chemists of Serbia
C3  - Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia
T1  - Protoplast patch-clamping using an upright microscope with a movable stage
SP  - 271
EP  - 274
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5508
ER  - 

@conference{
editor = "Čupić, Željko, Anić, Slobodan",
author = "Nikolić, Ljiljana and Vidović, Marija and Todorović, Nataša and Petković, Branka and Stojadinović, Gordana and Martać, Ljiljana and Bogdanović Pristov, Jelena",
year = "2022",
abstract = "Investigating membrane properties of plants is a challenging task, considering that success of
experiments is highly dependent on the possibility to isolate metabolically active protoplasts that
can withstand membrane current recordings. The aim of the present work is to obtain viable
protoplasts derived from root cells of Pisum sativum that can be used for the whole-cell patchclamp. We designed the procedure of the pea protoplasts isolation that delivers stable protoplasts
with preserved membrane integrity suitable for electrophysiological experiments. We applied a
custom approach for patch-clamping protoplasts using a microscope with a movable microscope
stage. We recorded prominent inward and prominent outward types of membrane current profiles of
protoplasts. Obtained data indicate that optimized isolation protocol and custom system for patchclamping, can be applied to study membrane properties of root protoplasts.",
publisher = "Belgrade: Society of Physical Chemists of Serbia",
journal = "Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia",
title = "Protoplast patch-clamping using an upright microscope with a movable stage",
pages = "271-274",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5508"
}

Čupić, Ž., Anić, S., Nikolić, L., Vidović, M., Todorović, N., Petković, B., Stojadinović, G., Martać, L.,& Bogdanović Pristov, J.. (2022). Protoplast patch-clamping using an upright microscope with a movable stage. in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia
Belgrade: Society of Physical Chemists of Serbia., 271-274.
https://hdl.handle.net/21.15107/rcub_ibiss_5508

Čupić Ž, Anić S, Nikolić L, Vidović M, Todorović N, Petković B, Stojadinović G, Martać L, Bogdanović Pristov J. Protoplast patch-clamping using an upright microscope with a movable stage. in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia. 2022;:271-274.
https://hdl.handle.net/21.15107/rcub_ibiss_5508 .

Čupić, Željko, Anić, Slobodan, Nikolić, Ljiljana, Vidović, Marija, Todorović, Nataša, Petković, Branka, Stojadinović, Gordana, Martać, Ljiljana, Bogdanović Pristov, Jelena, "Protoplast patch-clamping using an upright microscope with a movable stage" in Proceedings: Physical Chemistry 2022, Vol. 1.: 16th International Conference on Fundamental and Applied Aspects of Physical Chemistry; 2022 Sep 26-30; Belgrade, Serbia (2022):271-274,
https://hdl.handle.net/21.15107/rcub_ibiss_5508 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Rabasović, Mihailo D.
PY  - 2021
UR  - https://iupab2020.sbbq.org.br/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4719
AB  - Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.
PB  - São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular
C3  - 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
T1  - In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities
SP  - 358
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4719
ER  - 

@conference{
author = "Pajić, Tanja and Krmpot, Aleksandar J. and Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Rabasović, Mihailo D.",
year = "2021",
abstract = "Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.",
publisher = "São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular",
journal = "20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq",
title = "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities",
pages = "358",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4719"
}

Pajić, T., Krmpot, A. J., Todorović, N., Živić, M., Stevanović, K.,& Rabasović, M. D.. (2021). In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular., 358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719

Pajić T, Krmpot AJ, Todorović N, Živić M, Stevanović K, Rabasović MD. In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq. 2021;:358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

Pajić, Tanja, Krmpot, Aleksandar J., Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Rabasović, Mihailo D., "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities" in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq (2021):358,
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2021
UR  - https://iupab2020.sbbq.org.br/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4710
AB  - Outwardly rectifying, inactivating current (ORIC), recorded in cytoplasmatic droplets of P.blakesleeanus is 
characterized by a mild outward rectification, selectivity for anions, and a characteristic inactivation on 
depolarizing potentials. In absence of ATP, ORIC has a fast rundown with increasing speed of inactivation, while both processes are delayed by ATPpip (for at least 8min) and the non-hydrolysable AM-PCP (minimum of 5min). Extracellularly applied, ATP has a blocking effect. Flavonoid querzetin leads to a gradual loss of current despite the presence of intracellular ATP. Our research group has shown azide to be the potent respiration inhibitor of P. blakesleeanus, leaving the cells viable but significantly depleted from ATP. We are presenting data recorded in whole cell configuration, showing that: 1.ORIC can be activated despite the depletion of ATP in the cytoplasmatic droplet. However, its inactivation profile is significantly different comparing to normal conditions with or without ATP in the pipette in the first minute of recording, with reduced τ on lower depolarizing potentials. The fraction of current blocked by querzetin with AM-PCP in pipette solution is greater comparing to experiments with ATPpip, even with the 5-fold increase of AM-PCPpip concentration. The time required for current to recover from depolarizing steps is shorter at more hyperpolarized potentials. We have also started to look for the first insights into ORIC single channel activity in out-out configuration, and recorded, upon stimulation of ORIC in whole cell, and patch excision, the step-like pattern of inactivation that strongly resembles one of ORIC. Based on current data, we can conclude that ATP has a significant role in the inactivation process, with a more potent effect comparing to AM-PCP, whether by the means of greater affinity for the binding site or an additional mechanism involving kinase activity.
PB  - São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular
C3  - 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
T1  - Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior
SP  - 143
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4710
ER  - 

@conference{
author = "Stevanović, Katarina and Živić, Miroslav and Todorović, Nataša",
year = "2021",
abstract = "Outwardly rectifying, inactivating current (ORIC), recorded in cytoplasmatic droplets of P.blakesleeanus is 
characterized by a mild outward rectification, selectivity for anions, and a characteristic inactivation on 
depolarizing potentials. In absence of ATP, ORIC has a fast rundown with increasing speed of inactivation, while both processes are delayed by ATPpip (for at least 8min) and the non-hydrolysable AM-PCP (minimum of 5min). Extracellularly applied, ATP has a blocking effect. Flavonoid querzetin leads to a gradual loss of current despite the presence of intracellular ATP. Our research group has shown azide to be the potent respiration inhibitor of P. blakesleeanus, leaving the cells viable but significantly depleted from ATP. We are presenting data recorded in whole cell configuration, showing that: 1.ORIC can be activated despite the depletion of ATP in the cytoplasmatic droplet. However, its inactivation profile is significantly different comparing to normal conditions with or without ATP in the pipette in the first minute of recording, with reduced τ on lower depolarizing potentials. The fraction of current blocked by querzetin with AM-PCP in pipette solution is greater comparing to experiments with ATPpip, even with the 5-fold increase of AM-PCPpip concentration. The time required for current to recover from depolarizing steps is shorter at more hyperpolarized potentials. We have also started to look for the first insights into ORIC single channel activity in out-out configuration, and recorded, upon stimulation of ORIC in whole cell, and patch excision, the step-like pattern of inactivation that strongly resembles one of ORIC. Based on current data, we can conclude that ATP has a significant role in the inactivation process, with a more potent effect comparing to AM-PCP, whether by the means of greater affinity for the binding site or an additional mechanism involving kinase activity.",
publisher = "São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular",
journal = "20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq",
title = "Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior",
pages = "143",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4710"
}

Stevanović, K., Živić, M.,& Todorović, N.. (2021). Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular., 143.
https://hdl.handle.net/21.15107/rcub_ibiss_4710

Stevanović K, Živić M, Todorović N. Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq. 2021;:143.
https://hdl.handle.net/21.15107/rcub_ibiss_4710 .

Stevanović, Katarina, Živić, Miroslav, Todorović, Nataša, "Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior" in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq (2021):143,
https://hdl.handle.net/21.15107/rcub_ibiss_4710 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
AU  - Clayton, Andrew H. A.
AU  - Krmpot, Aleksandar J.
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4712
AB  - Oleaginous fungi can accumulate significant amounts of lipids in their mycelium (up to 80% of their biomass), primarily in the form of lipid droplets (LDs). LDs have optical properties that differ from the surrounding aquatic environment, which causes sudden changes in the refractive index. Here, we present in vivo and label-free imaging of individual hyphae of the oleaginous filamentous fungus 
Phycomyces blakesleeanus by Third Harmonic Generation (THG) microscopy method, where LDs 
are the main source of contrast. The LDs quantification from THG images was performed by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting – Particle Size Analysis (PSA). ICS measures the spatial variation of fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state. In order to test and compare the two methods, we used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and the increase in LDs number. For in vivo THG imaging of label-free, > 24-hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. Detection was performed in the transmission arm by PMT through Hoya glass UV filter with peak transmission at 340nm. The laser beam was focused with the Zeiss Plan Neofluar 40x1.3 objective lens. For the imaging, the fungi were placed between two cover glasses of 0.17 μm thickness to match the objective lens requirements and for better transmission of the THG signal. An increased number of LDs under nitrogen starvation was observed in THG images and their 
number and size were analyzed using two quantification methods. The comparison of LDs number and 
size obtained by ICS and PSA shows that the number of LDs is approximately the same on average, but 
that ICS consistently detects slightly larger LD number in older group. The mean ICS measured diameter was slightly lower. Using the THG method in vivo and label-free, we can accurately and reliably, over time, detect changes in the localization, total number, and size of LDs in hyphae of the oleaginous filamentous fungus Phycomyces blakesleeanus.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi
SP  - 115
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4712
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Rabasović, Mihailo D. and Clayton, Andrew H. A. and Krmpot, Aleksandar J.",
year = "2021",
abstract = "Oleaginous fungi can accumulate significant amounts of lipids in their mycelium (up to 80% of their biomass), primarily in the form of lipid droplets (LDs). LDs have optical properties that differ from the surrounding aquatic environment, which causes sudden changes in the refractive index. Here, we present in vivo and label-free imaging of individual hyphae of the oleaginous filamentous fungus 
Phycomyces blakesleeanus by Third Harmonic Generation (THG) microscopy method, where LDs 
are the main source of contrast. The LDs quantification from THG images was performed by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting – Particle Size Analysis (PSA). ICS measures the spatial variation of fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state. In order to test and compare the two methods, we used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and the increase in LDs number. For in vivo THG imaging of label-free, > 24-hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. Detection was performed in the transmission arm by PMT through Hoya glass UV filter with peak transmission at 340nm. The laser beam was focused with the Zeiss Plan Neofluar 40x1.3 objective lens. For the imaging, the fungi were placed between two cover glasses of 0.17 μm thickness to match the objective lens requirements and for better transmission of the THG signal. An increased number of LDs under nitrogen starvation was observed in THG images and their 
number and size were analyzed using two quantification methods. The comparison of LDs number and 
size obtained by ICS and PSA shows that the number of LDs is approximately the same on average, but 
that ICS consistently detects slightly larger LD number in older group. The mean ICS measured diameter was slightly lower. Using the THG method in vivo and label-free, we can accurately and reliably, over time, detect changes in the localization, total number, and size of LDs in hyphae of the oleaginous filamentous fungus Phycomyces blakesleeanus.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi",
pages = "115",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4712"
}

Pajić, T., Todorović, N., Živić, M., Rabasović, M. D., Clayton, A. H. A.,& Krmpot, A. J.. (2021). Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 115.
https://hdl.handle.net/21.15107/rcub_ibiss_4712

Pajić T, Todorović N, Živić M, Rabasović MD, Clayton AHA, Krmpot AJ. Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:115.
https://hdl.handle.net/21.15107/rcub_ibiss_4712 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Rabasović, Mihailo D., Clayton, Andrew H. A., Krmpot, Aleksandar J., "Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):115,
https://hdl.handle.net/21.15107/rcub_ibiss_4712 .

TY  - CONF
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4713
AB  - Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery
SP  - 111
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4713
ER  - 

@conference{
author = "Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Todorović, Nataša",
year = "2021",
abstract = "Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery",
pages = "111",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4713"
}

Živić, M., Stevanović, K., Pajić, T., Rabasović, M. D., Krmpot, A. J.,& Todorović, N.. (2021). First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713

Živić M, Stevanović K, Pajić T, Rabasović MD, Krmpot AJ, Todorović N. First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Rabasović, Mihailo D., Krmpot, Aleksandar J., Todorović, Nataša, "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):111,
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4722
AB  - Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to 
test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, 
induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium 
azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data 
obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and 
the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small 
confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and 
in smaller confinement than controls.
PB  - Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac
C3  - 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
T1  - The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor
DO  - 10.46793/ICCBI21.226P
SP  - 226
EP  - 229
ER  - 

@conference{
author = "Pajić, Tanja and Živić, Miroslav and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Todorović, Nataša",
year = "2021",
abstract = "Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to 
test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, 
induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium 
azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data 
obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and 
the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small 
confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and 
in smaller confinement than controls.",
publisher = "Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac",
journal = "1st International Conference on Chemo and BioInformatics, ICCBIKG 2021",
title = "The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor",
doi = "10.46793/ICCBI21.226P",
pages = "226-229"
}

Pajić, T., Živić, M., Rabasović, M. D., Krmpot, A. J.,& Todorović, N.. (2021). The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac., 226-229.
https://doi.org/10.46793/ICCBI21.226P

Pajić T, Živić M, Rabasović MD, Krmpot AJ, Todorović N. The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021. 2021;:226-229.
doi:10.46793/ICCBI21.226P .

Pajić, Tanja, Živić, Miroslav, Rabasović, Mihailo D., Krmpot, Aleksandar J., Todorović, Nataša, "The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor" in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021 (2021):226-229,
https://doi.org/10.46793/ICCBI21.226P . .

TY  - CONF
AU  - AlJamal, Kamal
AU  - Stojadinović, Gordana
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4711
AB  - The chloride channel VRAC has been implicated in apoptosis, proliferation, transport of organic acids and regulatory volume decrease. Regulated by RAS/cGMP and by the ionic strength, it mediates efflux of anions (Cl-, glutamate, gluthatione, taurine, cGAMP). Knowing that chloride transport activity, mediated by NKCC, is indispensable for axon growth, we hypothesized that VRAC could have a role in neurite elongation mechanism. Experimental treatments compared to control were: VRAC selective inhibitor DCPIB (10 µM); BpV (phen) (80 nM), an inhibitor of PTEN. Axon growth was measured on cultured 
dissociated dorsal root ganglia neonatal neurons fixed 24h after treatment and immunostained with anti-neurofilament H (NF-H) phosphorylated antibody. FIJI Simple Neurite Tracer was used for morphometry of individual neurons. 24h post treatment, total neurite length compared to control was decreased by BpV (p=0.0058) and to a lesser extent by DCPIB. In all measured parameters related to the degree of branching, BpV group neurons had small dispersion of values. In addition, DCPIB treated cells had 
larger fractal dimension (<0.05) than control neurons. The effect of DCPIB is mostly related to an increase in Fractal Dimension parameter, according to Principal Component Analysis. The increase in the number of primary branches per increment of cable length is significantly smaller in DCPIB group than in control and BpV -treated neurons. The DCPIB effect demonstrates that an anion current, presumably VRAC, has 
a role in the process of proper branching and directional elongation of neonatal DRG neurites.
C3  - EMBO Workshop Axons 2021: Structure and function
T1  - Morphometric comparison of neurite outgrowth in rat DRG cultures treated with DCPIB and BpV
SP  - 74
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4711
ER  - 

@conference{
author = "AlJamal, Kamal and Stojadinović, Gordana and Živić, Miroslav and Todorović, Nataša",
year = "2021",
abstract = "The chloride channel VRAC has been implicated in apoptosis, proliferation, transport of organic acids and regulatory volume decrease. Regulated by RAS/cGMP and by the ionic strength, it mediates efflux of anions (Cl-, glutamate, gluthatione, taurine, cGAMP). Knowing that chloride transport activity, mediated by NKCC, is indispensable for axon growth, we hypothesized that VRAC could have a role in neurite elongation mechanism. Experimental treatments compared to control were: VRAC selective inhibitor DCPIB (10 µM); BpV (phen) (80 nM), an inhibitor of PTEN. Axon growth was measured on cultured 
dissociated dorsal root ganglia neonatal neurons fixed 24h after treatment and immunostained with anti-neurofilament H (NF-H) phosphorylated antibody. FIJI Simple Neurite Tracer was used for morphometry of individual neurons. 24h post treatment, total neurite length compared to control was decreased by BpV (p=0.0058) and to a lesser extent by DCPIB. In all measured parameters related to the degree of branching, BpV group neurons had small dispersion of values. In addition, DCPIB treated cells had 
larger fractal dimension (<0.05) than control neurons. The effect of DCPIB is mostly related to an increase in Fractal Dimension parameter, according to Principal Component Analysis. The increase in the number of primary branches per increment of cable length is significantly smaller in DCPIB group than in control and BpV -treated neurons. The DCPIB effect demonstrates that an anion current, presumably VRAC, has 
a role in the process of proper branching and directional elongation of neonatal DRG neurites.",
journal = "EMBO Workshop Axons 2021: Structure and function",
title = "Morphometric comparison of neurite outgrowth in rat DRG cultures treated with DCPIB and BpV",
pages = "74",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4711"
}

AlJamal, K., Stojadinović, G., Živić, M.,& Todorović, N.. (2021). Morphometric comparison of neurite outgrowth in rat DRG cultures treated with DCPIB and BpV. in EMBO Workshop Axons 2021: Structure and function, 74.
https://hdl.handle.net/21.15107/rcub_ibiss_4711

AlJamal K, Stojadinović G, Živić M, Todorović N. Morphometric comparison of neurite outgrowth in rat DRG cultures treated with DCPIB and BpV. in EMBO Workshop Axons 2021: Structure and function. 2021;:74.
https://hdl.handle.net/21.15107/rcub_ibiss_4711 .

AlJamal, Kamal, Stojadinović, Gordana, Živić, Miroslav, Todorović, Nataša, "Morphometric comparison of neurite outgrowth in rat DRG cultures treated with DCPIB and BpV" in EMBO Workshop Axons 2021: Structure and function (2021):74,
https://hdl.handle.net/21.15107/rcub_ibiss_4711 .