Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, gua... more Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, guarantees plasma membrane homeostasis, and generates intracellular signaling cascades. Moreover, it plays an important role in dead cell clearance and defense against external microbes. Finally, endocytosis is an important cellular route for the delivery of nanomedicines for therapeutic treatments. Thus, it is not surprising that both environmental and genetic perturbation of endocytosis have been associated with several human conditions such as cancer, neurological disorders, and virus infections, among others. Over the last decades, a lot of research has been focused on developing advanced imaging methods to monitor endocytosis events with high resolution in living cells and tissues. These include fluorescence imaging, electron microscopy, and correlative and super-resolution microscopy. In this review, we outline the major endocytic pathways and briefly discuss how defects in the molecu...
International Journal of Environmental Research and Public Health
Aluminum is an element found in nature and in cosmetic products. It can interfere with the metabo... more Aluminum is an element found in nature and in cosmetic products. It can interfere with the metabolism of other cations, thus inducing gastrointestinal disorder. In cosmetics, aluminum is used in antiperspirants, lipsticks, and toothpastes. The aim of this work is to investigate aluminum bioavailability after accidental oral ingestion derived from the use of a toothpaste containing a greater amount of aluminum hydroxide than advised by the Scientific Committee on Consumer Safety (SCCS). To simulate in vitro toothpaste accidental ingestion, the INFOGEST model was employed, and the amount of aluminum was measured through the ICP-AES analysis. Tissue barrier integrity was analyzed by measuring transepithelial electric resistance, and the tissue architecture was checked through light microscopy. The margin of safety was also calculated. Overall, our results indicate that the acute exposure to aluminum accidentally ingested from toothpastes is safe for the final user, even in amounts high...
Melanocytes represent the second most important cell type in the skin and are primarily responsib... more Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.
CA and Tz up-regulate p21WAF1 expression levels in SKBR-3 and BT474 cells. Cells were cultured fo... more CA and Tz up-regulate p21WAF1 expression levels in SKBR-3 and BT474 cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. A representative immunoblot analysis is shown, which was performed with an anti-p21WAF1 antibody on whole cell lysates. Tubulin is shown as loading control. Numbers on each lane represent p21WAF1 protein levels determined as described in Methods. (TIFF 222 kb)
We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Baris... more We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Barisione et al. 2020 doi.org/10.1007/s00428-020-02934-1) by using thin section electron microscopy. A detailed description of the methods and the data set is provided in the download container. Data set 13 contains stitched image montages of a thin section through the lung of patient C03 which were acquired by scanning electron microscopy. The file "Data_set_13.tif" contains a montage of the entire thin section while the other files contain selected areas of the section recorded at higher resolution.
We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Baris... more We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Barisione et al. 2020 doi.org/10.1007/s00428-020-02934-1) by using thin section electron microscopy. A detailed description of the methods and the data set is provided in the download container. Data set 09 contains stitched image montages of thin sections (selected areas) through the lung of patient C03 which were acquired by scanning electron microscopy (C03_A & C) or transmission electron microscopy (C03_B). The images show accumulation of cells and debris in the alveolar cavity.
Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Tr... more Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (...
Ki-67 expression modulation by CA and Tz. Cells were cultured for 7d with control medium (Ctr), C... more Ki-67 expression modulation by CA and Tz. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. Cells were fixed and permeabilized and Ki-67 was detected by indirect immunofluorescence analysis using a mouse anti-Ki-67 antibody (see Additional file 1: Table S1) and Alexa488-conjugated goat anti-mouse antibodies. Nuclei were stained with DAPI. Ki-67 positive nuclei and total nuclei were counted in blind. Four microscopic fields (n = 4) were analyzed for each experimental condition. Mean values and standard deviation (indicated as vertical bars) are shown. P
Representative DNA content histograms obtained by high resolution DNA flow cytometry from SKBR-3 ... more Representative DNA content histograms obtained by high resolution DNA flow cytometry from SKBR-3 and BT474 cells, top and bottom row respectively. X axes show DNA content measured as intensity of fluorescent light emitted by DNA bound DAPI at 435 nm; Y axes show number of nuclei (counts). Arrow heads indicate G0/G1 and G2/M peaks. Control treatment, Ctr; Carnosic acid treatment; CA; Trastuzumab treatment, Tz; Carnosic plus Trastuzumab treatment, CA + Tz. (TIFF 396 kb)
CA impairs survival, increases P21WAF1 and p62 expression levels and deranges the lysosomal compa... more CA impairs survival, increases P21WAF1 and p62 expression levels and deranges the lysosomal compartment of MCF10A cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. (A) Cell survival is expressed as arbitrary units (A.U.) after exposure to MTT agent for 4 h. Mean values and standard deviation (indicated as vertical bars) from three independent replicates (n = 3) are shown. P
CA inhibits ERBB2− in vitro breast cancer cell survival and migration. MCF7 and MDA-MB-231 cells ... more CA inhibits ERBB2− in vitro breast cancer cell survival and migration. MCF7 and MDA-MB-231 cells were cultured for 10 days with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. a Cell survival is expressed as arbitrary units (A.U.) after exposure to MTT agent for 4 h. Polynomial (Poly.) interpolation curves are shown. Mean values and standard deviation (indicated as vertical bars) from three independent replicates are shown. b MDA-MB-231 cells were cultured after overnight starvation for 7d with control medium (Ctr) or CA supplemented medium in the lower migration chamber, which was changed every 48 h. Representative images of the lower side of the migration membranes stained with Crystal Violet. Bar = 100 μm. c Relative cell migration expressed as percentage (%) compared to control was determined as detailed in Methods section. Mean values and standard deviation (indicated as vertical bars) from four independent replicates (n = 4) are shown. P
Analysis of adipogenic markers expression in ERBB2+ BC cells. Cells were cultured for 7d with con... more Analysis of adipogenic markers expression in ERBB2+ BC cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. A) PLIN1 and PLIN2 mRNA expression levels were detect by RT qPCR analysis and are expressed as arbitrary units (A.U.). Mean values and standard deviation (indicated as vertical bars) from three independent replicates (n = 3) are shown. P
Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they m... more Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuron...
Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, gua... more Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, guarantees plasma membrane homeostasis, and generates intracellular signaling cascades. Moreover, it plays an important role in dead cell clearance and defense against external microbes. Finally, endocytosis is an important cellular route for the delivery of nanomedicines for therapeutic treatments. Thus, it is not surprising that both environmental and genetic perturbation of endocytosis have been associated with several human conditions such as cancer, neurological disorders, and virus infections, among others. Over the last decades, a lot of research has been focused on developing advanced imaging methods to monitor endocytosis events with high resolution in living cells and tissues. These include fluorescence imaging, electron microscopy, and correlative and super-resolution microscopy. In this review, we outline the major endocytic pathways and briefly discuss how defects in the molecu...
International Journal of Environmental Research and Public Health
Aluminum is an element found in nature and in cosmetic products. It can interfere with the metabo... more Aluminum is an element found in nature and in cosmetic products. It can interfere with the metabolism of other cations, thus inducing gastrointestinal disorder. In cosmetics, aluminum is used in antiperspirants, lipsticks, and toothpastes. The aim of this work is to investigate aluminum bioavailability after accidental oral ingestion derived from the use of a toothpaste containing a greater amount of aluminum hydroxide than advised by the Scientific Committee on Consumer Safety (SCCS). To simulate in vitro toothpaste accidental ingestion, the INFOGEST model was employed, and the amount of aluminum was measured through the ICP-AES analysis. Tissue barrier integrity was analyzed by measuring transepithelial electric resistance, and the tissue architecture was checked through light microscopy. The margin of safety was also calculated. Overall, our results indicate that the acute exposure to aluminum accidentally ingested from toothpastes is safe for the final user, even in amounts high...
Melanocytes represent the second most important cell type in the skin and are primarily responsib... more Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.
CA and Tz up-regulate p21WAF1 expression levels in SKBR-3 and BT474 cells. Cells were cultured fo... more CA and Tz up-regulate p21WAF1 expression levels in SKBR-3 and BT474 cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. A representative immunoblot analysis is shown, which was performed with an anti-p21WAF1 antibody on whole cell lysates. Tubulin is shown as loading control. Numbers on each lane represent p21WAF1 protein levels determined as described in Methods. (TIFF 222 kb)
We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Baris... more We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Barisione et al. 2020 doi.org/10.1007/s00428-020-02934-1) by using thin section electron microscopy. A detailed description of the methods and the data set is provided in the download container. Data set 13 contains stitched image montages of a thin section through the lung of patient C03 which were acquired by scanning electron microscopy. The file "Data_set_13.tif" contains a montage of the entire thin section while the other files contain selected areas of the section recorded at higher resolution.
We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Baris... more We investigated six cryobiopsy samples from six deceased patients (patients C03 to C08 from Barisione et al. 2020 doi.org/10.1007/s00428-020-02934-1) by using thin section electron microscopy. A detailed description of the methods and the data set is provided in the download container. Data set 09 contains stitched image montages of thin sections (selected areas) through the lung of patient C03 which were acquired by scanning electron microscopy (C03_A & C) or transmission electron microscopy (C03_B). The images show accumulation of cells and debris in the alveolar cavity.
Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Tr... more Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is an ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles (EVs) released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here, we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Trastuzumab (...
Ki-67 expression modulation by CA and Tz. Cells were cultured for 7d with control medium (Ctr), C... more Ki-67 expression modulation by CA and Tz. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. Cells were fixed and permeabilized and Ki-67 was detected by indirect immunofluorescence analysis using a mouse anti-Ki-67 antibody (see Additional file 1: Table S1) and Alexa488-conjugated goat anti-mouse antibodies. Nuclei were stained with DAPI. Ki-67 positive nuclei and total nuclei were counted in blind. Four microscopic fields (n = 4) were analyzed for each experimental condition. Mean values and standard deviation (indicated as vertical bars) are shown. P
Representative DNA content histograms obtained by high resolution DNA flow cytometry from SKBR-3 ... more Representative DNA content histograms obtained by high resolution DNA flow cytometry from SKBR-3 and BT474 cells, top and bottom row respectively. X axes show DNA content measured as intensity of fluorescent light emitted by DNA bound DAPI at 435 nm; Y axes show number of nuclei (counts). Arrow heads indicate G0/G1 and G2/M peaks. Control treatment, Ctr; Carnosic acid treatment; CA; Trastuzumab treatment, Tz; Carnosic plus Trastuzumab treatment, CA + Tz. (TIFF 396 kb)
CA impairs survival, increases P21WAF1 and p62 expression levels and deranges the lysosomal compa... more CA impairs survival, increases P21WAF1 and p62 expression levels and deranges the lysosomal compartment of MCF10A cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. (A) Cell survival is expressed as arbitrary units (A.U.) after exposure to MTT agent for 4 h. Mean values and standard deviation (indicated as vertical bars) from three independent replicates (n = 3) are shown. P
CA inhibits ERBB2− in vitro breast cancer cell survival and migration. MCF7 and MDA-MB-231 cells ... more CA inhibits ERBB2− in vitro breast cancer cell survival and migration. MCF7 and MDA-MB-231 cells were cultured for 10 days with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. a Cell survival is expressed as arbitrary units (A.U.) after exposure to MTT agent for 4 h. Polynomial (Poly.) interpolation curves are shown. Mean values and standard deviation (indicated as vertical bars) from three independent replicates are shown. b MDA-MB-231 cells were cultured after overnight starvation for 7d with control medium (Ctr) or CA supplemented medium in the lower migration chamber, which was changed every 48 h. Representative images of the lower side of the migration membranes stained with Crystal Violet. Bar = 100 μm. c Relative cell migration expressed as percentage (%) compared to control was determined as detailed in Methods section. Mean values and standard deviation (indicated as vertical bars) from four independent replicates (n = 4) are shown. P
Analysis of adipogenic markers expression in ERBB2+ BC cells. Cells were cultured for 7d with con... more Analysis of adipogenic markers expression in ERBB2+ BC cells. Cells were cultured for 7d with control medium (Ctr), CA, Tz or CA + Tz supplemented medium, which was changed every 48 h. A) PLIN1 and PLIN2 mRNA expression levels were detect by RT qPCR analysis and are expressed as arbitrary units (A.U.). Mean values and standard deviation (indicated as vertical bars) from three independent replicates (n = 3) are shown. P
Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they m... more Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuron...
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