Kosala D. Waduthanthri, C. Montemagno, Sibel Çetinel
Human trabecular meshwork (hTM) cell isolation in academic settings utilizes the motile nature of these cells, allowing them to migrate away from the explant and proliferate on distal regions of the culture substrate. Corneoscleral rims used for transplantation are a potential source of explants for the establishment of hTM cell cultures. However, cell isolation and the initiation of primary cell cultures from ocular tissues stored in Optisol-GS medium for an extended period of time (>6 days) has proven difficult, since Optisol-GS remarkably reduces cell viability and cellularity. Therefore, explants obtained from ocular tissues stored in Optisol-GS do not often provide adequate cell yield to initiate primary cell cultures if conventional culture techniques are used. Therefore, the majority of the research on primary hTM cell isolation has been accomplished using donor tissue obtained within 72 h postmortem. The goal of this study was to develop an hTM cell isolation procedure from nontransplantable ocular materials, utilizing the anchorage dependency of TM cells. This procedure yielded functionally viable cells, eficiently dissociated from the trabecular meshwork. Isolated cells demonstrated typical hTM cell characteristics including monolayer formation, contact inhibition, phagocytosis, and responses to glucocorticoid exposure. To the best of our knowledge, this is the first time an expired explant has been utilized in the successful isolation of hTM cells. Our results clearly demonstrate the advantage of increasing the anchor points of hTM cells for enhanced cell migration out from the explants, which have limited cell proliferative capacity.
{"title":"Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims","authors":"Kosala D. Waduthanthri, C. Montemagno, Sibel Çetinel","doi":"10.3906/BIY-1810-69","DOIUrl":"https://doi.org/10.3906/BIY-1810-69","url":null,"abstract":"Human trabecular meshwork (hTM) cell isolation in academic settings utilizes the motile nature of these cells, allowing them to migrate away from the explant and proliferate on distal regions of the culture substrate. Corneoscleral rims used for transplantation are a potential source of explants for the establishment of hTM cell cultures. However, cell isolation and the initiation of primary cell cultures from ocular tissues stored in Optisol-GS medium for an extended period of time (>6 days) has proven difficult, since Optisol-GS remarkably reduces cell viability and cellularity. Therefore, explants obtained from ocular tissues stored in Optisol-GS do not often provide adequate cell yield to initiate primary cell cultures if conventional culture techniques are used. Therefore, the majority of the research on primary hTM cell isolation has been accomplished using donor tissue obtained within 72 h postmortem. The goal of this study was to develop an hTM cell isolation procedure from nontransplantable ocular materials, utilizing the anchorage dependency of TM cells. This procedure yielded functionally viable cells, eficiently dissociated from the trabecular meshwork. Isolated cells demonstrated typical hTM cell characteristics including monolayer formation, contact inhibition, phagocytosis, and responses to glucocorticoid exposure. To the best of our knowledge, this is the first time an expired explant has been utilized in the successful isolation of hTM cells. Our results clearly demonstrate the advantage of increasing the anchor points of hTM cells for enhanced cell migration out from the explants, which have limited cell proliferative capacity.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"43 1","pages":"89 - 98"},"PeriodicalIF":2.2,"publicationDate":"2019-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1810-69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42218700","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shigellosis is one of the most important acute enteric infections caused by different species of Shigella, such as Shigella flexneri. Despite the use of antibiotic therapy to reduce disease duration, this approach is becoming less effective due to the emergence of antibiotic resistance among Shigella spp. Bacteriophages have been introduced as an alternative for controlling shigellosis. However, the bacteriophages must be without any lysogenic or virulence factors, toxin coding, or antibiotic-resistant genes. In this study, the whole genome sequence of vB-SflS-ISF001, a virulent Siphoviridae bacteriophage specific for Shigella flexneri, was obtained, and a comparative genomic analysis was carried out to identify its properties and safety. vB-SflS-ISF001 genomic DNA was measured at 50,552 bp with 78 deduced open reading frames (ORFs), with 24 ORFs (30.77%) sharing similarities with proteins from the genomes of homologous phages that had been reported earlier. Genetic analysis classifies it under the genus T1virus of the subfamily Tunavirinae . Moreover, comparative genomic analysis revealed no undesirable genes in the genome of vB-SflS-ISF001, such as antibiotic resistance, virulence, lysogeny, or toxin-coding genes. The results of this investigation indicate that vB-SflS-ISF001 is a new species, and confirm its safety for the biocontrol of S. flexneri.
{"title":"Complete genome sequence analysis of a lytic Shigella flexneri vB-SflS-ISF001 bacteriophage","authors":"Khashayar Shahin, M. Bouzari, Ran Wang","doi":"10.3906/BIY-1808-97","DOIUrl":"https://doi.org/10.3906/BIY-1808-97","url":null,"abstract":"Shigellosis is one of the most important acute enteric infections caused by different species of Shigella, such as Shigella flexneri. Despite the use of antibiotic therapy to reduce disease duration, this approach is becoming less effective due to the emergence of antibiotic resistance among Shigella spp. Bacteriophages have been introduced as an alternative for controlling shigellosis. However, the bacteriophages must be without any lysogenic or virulence factors, toxin coding, or antibiotic-resistant genes. In this study, the whole genome sequence of vB-SflS-ISF001, a virulent Siphoviridae bacteriophage specific for Shigella flexneri, was obtained, and a comparative genomic analysis was carried out to identify its properties and safety. vB-SflS-ISF001 genomic DNA was measured at 50,552 bp with 78 deduced open reading frames (ORFs), with 24 ORFs (30.77%) sharing similarities with proteins from the genomes of homologous phages that had been reported earlier. Genetic analysis classifies it under the genus T1virus of the subfamily Tunavirinae . Moreover, comparative genomic analysis revealed no undesirable genes in the genome of vB-SflS-ISF001, such as antibiotic resistance, virulence, lysogeny, or toxin-coding genes. The results of this investigation indicate that vB-SflS-ISF001 is a new species, and confirm its safety for the biocontrol of S. flexneri.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"43 1","pages":"99 - 112"},"PeriodicalIF":2.2,"publicationDate":"2019-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1808-97","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42321357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transformed follicular lymphoma (tFL) originates from histological transformation of follicular lymphoma (FL), which is the most common indolent non-Hodgkin lymphoma. High-resolution genomic copy-number analysis previously identified frequent amplification of the 2p15-p16.1 locus in FL and tFL cases. The genes (i.e. BCL11A, PAPOLG, PUS10, and USP34) in this amplified locus have not been systematically investigated to date in terms of their role in FL pathogenesis or transformation to tFL. Here we investigated the relationship between amplification and expression of genes in 2p15-p16.1 as well as their expression after histological transformation. NCBI GEO SNP array and gene expression profile (GEP) data of tFL cases were analyzed to evaluate the relationship between amplification and mRNA expression. Moreover, transcript levels of these four genes in FL cases were compared with those of patient-matched tFL cases and normal B-cells. Amplification of the 2p15-p16.1 locus is associated with increased transcription of BCL11A and PAPOLG in tFL cases, of which the latter showed increased expression after histological transformation. Compared with the level in normal B-cells, PAPOLG was significantly overexpressed in FL cases, but expression levels of the other three genes did not show any significant difference. Altogether these results suggest that PAPOLG may be the most critical gene in terms of transformation to tFL.
{"title":"Systematic analysis of the frequently amplified 2p15-p16.1 locus reveals PAPOLG as a potential proto-oncogene in follicular and transformed follicular lymphoma","authors":"Deniz Kurşun, Can Küçük","doi":"10.3906/biy-1810-2","DOIUrl":"https://doi.org/10.3906/biy-1810-2","url":null,"abstract":"Transformed follicular lymphoma (tFL) originates from histological transformation of follicular lymphoma (FL), which is the most common indolent non-Hodgkin lymphoma. High-resolution genomic copy-number analysis previously identified frequent amplification of the 2p15-p16.1 locus in FL and tFL cases. The genes (i.e. BCL11A, PAPOLG, PUS10, and USP34) in this amplified locus have not been systematically investigated to date in terms of their role in FL pathogenesis or transformation to tFL. Here we investigated the relationship between amplification and expression of genes in 2p15-p16.1 as well as their expression after histological transformation. NCBI GEO SNP array and gene expression profile (GEP) data of tFL cases were analyzed to evaluate the relationship between amplification and mRNA expression. Moreover, transcript levels of these four genes in FL cases were compared with those of patient-matched tFL cases and normal B-cells. Amplification of the 2p15-p16.1 locus is associated with increased transcription of BCL11A and PAPOLG in tFL cases, of which the latter showed increased expression after histological transformation. Compared with the level in normal B-cells, PAPOLG was significantly overexpressed in FL cases, but expression levels of the other three genes did not show any significant difference. Altogether these results suggest that PAPOLG may be the most critical gene in terms of transformation to tFL.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"43 1","pages":"124 - 132"},"PeriodicalIF":2.2,"publicationDate":"2019-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/biy-1810-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48954428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhiyuan Wei, Xiaohe Shen, B. Ni, G. Luo, Yi Tian, Yi Sun
The hepatitis B virus-encoded X (HBX) protein plays important roles in Hepatocellular carcinoma (HCC). Previous studies have demonstrated that HBX can induce alterations in the expression of numerous microRNAs (miRNAs) involved in the carcinogenesis of various tumors. However, the global profile of liver miRNA changes induced by HBX has not been characterized. In this study, we conducted a miRNA microarray analysis to investigate the influence of HBX on the expression of total miRNAs in liver in relation to HCC. Comparative analysis of the data from human normal liver cells (L02) and human HCC cells (HepG2), with or without HBX, identified 19 differentially expressed miRNAs, including 5 with known association to HBX. Target gene prediction for the aberrantly expressed miRNAs identified a total of 304 potential target genes, involved in sundry pathways. Finally, pathway analysis of the HBXinduced miRNAs pathway showed that 5 of the total miRNAs formed an internetwork, suggesting that HBX might exert its pathological effects on hepatic cells through functional synergy with miRNAs that regulated common pathways in liver cells. Therefore, this work provides new insights into the mechanisms of HCC as well as potential diagnostic markers or therapeutic targets for use in clinical management of HCC.
{"title":"Contribution of hepatitis B virus X protein-induced aberrant microRNA expression to hepatocellular carcinoma pathogenesis","authors":"Zhiyuan Wei, Xiaohe Shen, B. Ni, G. Luo, Yi Tian, Yi Sun","doi":"10.3906/biy-1807-196","DOIUrl":"https://doi.org/10.3906/biy-1807-196","url":null,"abstract":"The hepatitis B virus-encoded X (HBX) protein plays important roles in Hepatocellular carcinoma (HCC). Previous studies have demonstrated that HBX can induce alterations in the expression of numerous microRNAs (miRNAs) involved in the carcinogenesis of various tumors. However, the global profile of liver miRNA changes induced by HBX has not been characterized. In this study, we conducted a miRNA microarray analysis to investigate the influence of HBX on the expression of total miRNAs in liver in relation to HCC. Comparative analysis of the data from human normal liver cells (L02) and human HCC cells (HepG2), with or without HBX, identified 19 differentially expressed miRNAs, including 5 with known association to HBX. Target gene prediction for the aberrantly expressed miRNAs identified a total of 304 potential target genes, involved in sundry pathways. Finally, pathway analysis of the HBXinduced miRNAs pathway showed that 5 of the total miRNAs formed an internetwork, suggesting that HBX might exert its pathological effects on hepatic cells through functional synergy with miRNAs that regulated common pathways in liver cells. Therefore, this work provides new insights into the mechanisms of HCC as well as potential diagnostic markers or therapeutic targets for use in clinical management of HCC.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"43 1","pages":"113 - 123"},"PeriodicalIF":2.2,"publicationDate":"2019-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/biy-1807-196","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43329772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The aim of this research was to optimize the encapsulation-dehydration cryopreservation protocol of Lady Orange chrysanthemum explants on the preculture, pretreatment, and post-rewarming recovery steps. Shoot tips were precultured on MS medium with various abscisic acid concentrations (0-30 µM). Next, the encapsulated explants were osmotically dehydrated for 3 or 5 days and desiccated for 3-4 h and, after rewarming, they were subcultured on recovery media of various compositions (control and cytokinin/auxin-supplemented). A high explant survival rate, even up to 100%, was observed. The value of this parameter, however, changed depending on the post-rewarming culture duration. Moreover, not all the viable explants were capable of forming shoots. A lower ABA concentration (15 µM) during preculture and the presence of 4.65 µM kinetin in the post-rewarming recovery medium enhanced cryopreservation efficiency with a high survival rate and typical microshoot formation. A higher ABA concentration and the presence of 6-benzylaminopurine in the recovery medium resulted in shoot multiplication, abundant callus formation, and root formation inhibition.
{"title":"Effects of various preculture, pretreatment, and recovery conditions on the morphogenetic response of cryopreserved Lady Orange chrysanthemum shoot tips","authors":"D. Kulus","doi":"10.3906/BIY-1711-47","DOIUrl":"https://doi.org/10.3906/BIY-1711-47","url":null,"abstract":"The aim of this research was to optimize the encapsulation-dehydration cryopreservation protocol of Lady Orange chrysanthemum explants on the preculture, pretreatment, and post-rewarming recovery steps. Shoot tips were precultured on MS medium with various abscisic acid concentrations (0-30 µM). Next, the encapsulated explants were osmotically dehydrated for 3 or 5 days and desiccated for 3-4 h and, after rewarming, they were subcultured on recovery media of various compositions (control and cytokinin/auxin-supplemented). A high explant survival rate, even up to 100%, was observed. The value of this parameter, however, changed depending on the post-rewarming culture duration. Moreover, not all the viable explants were capable of forming shoots. A lower ABA concentration (15 µM) during preculture and the presence of 4.65 µM kinetin in the post-rewarming recovery medium enhanced cryopreservation efficiency with a high survival rate and typical microshoot formation. A higher ABA concentration and the presence of 6-benzylaminopurine in the recovery medium resulted in shoot multiplication, abundant callus formation, and root formation inhibition.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"42 1","pages":"76-86"},"PeriodicalIF":2.2,"publicationDate":"2018-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1711-47","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44327863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Comparative proteomic analysis of Bacillus thuringiensis wild-type and two mutant strains disturbed in polyphosphate homeostasis","authors":"Filiz Yesilirmak, Tuğrul Doruk, S. Yilmaz, Sedef Tunca Gedik","doi":"10.3906/BIY-1711-9","DOIUrl":"https://doi.org/10.3906/BIY-1711-9","url":null,"abstract":"The Scientific and Technological Research Council of Turkey [TBAG-107T812, TBAG-111T047, KBAG-113Z898]","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"42 1","pages":"87-102"},"PeriodicalIF":2.2,"publicationDate":"2018-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1711-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42374555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"In vitro transcription and validation of human pancreatic transcription factors' mRNAs","authors":"Ersin Akinci, M. Yildiz, P. Unal, Gamze Badakul","doi":"10.3906/BIY-1610-29","DOIUrl":"https://doi.org/10.3906/BIY-1610-29","url":null,"abstract":"* Correspondence: ersinakinci@akdeniz.edu.tr","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"708-718"},"PeriodicalIF":2.2,"publicationDate":"2017-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1610-29","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48364497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In the treatment of dermal wounds, wound-dressing materials prepared from natural mucopolysaccharides are widely used because of their advantages such as nonirritation, nontoxicity, and ease in topical application. In the present study, alginate hydrogels modified with N-acetyl glucose amine (NAG) were prepared as wound-dressing material. Physical, chemical, thermal, and mechanical properties of the hydrogels were studied. Cytotoxicity of the hydrogels on endothelial (HUVEC) and keratinocyte (HaCaT) cells were examined. Anti- and proinflammatory cytokine levels of human monocyte-macrophage cells (THP-1) stimulated with hydrogels were determined. According to the results, increasing the NAG concentration led to an increase in the swelling and nitrogen ratios in the hydrogels. Additionally, increasing the NAG concentration decreased elastic modulus and degradation time. Hydrogels were not cytotoxic on HaCaT and HUVEC cells. It stimulated IL-10 and TNF-alpha levels at a small rate.
{"title":"N-Acetylglucoseamine modified alginate sponges as scaffolds for skin tissue engineering","authors":"M. Demirbilek, N. Türkoğlu, S. Aktürk","doi":"10.3906/BIY-1704-31","DOIUrl":"https://doi.org/10.3906/BIY-1704-31","url":null,"abstract":"In the treatment of dermal wounds, wound-dressing materials prepared from natural mucopolysaccharides are widely used because of their advantages such as nonirritation, nontoxicity, and ease in topical application. In the present study, alginate hydrogels modified with N-acetyl glucose amine (NAG) were prepared as wound-dressing material. Physical, chemical, thermal, and mechanical properties of the hydrogels were studied. Cytotoxicity of the hydrogels on endothelial (HUVEC) and keratinocyte (HaCaT) cells were examined. Anti- and proinflammatory cytokine levels of human monocyte-macrophage cells (THP-1) stimulated with hydrogels were determined. According to the results, increasing the NAG concentration led to an increase in the swelling and nitrogen ratios in the hydrogels. Additionally, increasing the NAG concentration decreased elastic modulus and degradation time. Hydrogels were not cytotoxic on HaCaT and HUVEC cells. It stimulated IL-10 and TNF-alpha levels at a small rate.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"796-807"},"PeriodicalIF":2.2,"publicationDate":"2017-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1704-31","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47561654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mitochondrial dysfunction has been previously identified in neurodegenerative diseases such as Alzheimer disease, Huntington disease, and Parkinson disease. Chemical inhibition of the mitochondrial electron transport chain (ETC) was shown to trigger symptoms in animal models similar to those observed in human neurodegenerative diseases. In order to understand the effect of mitochondrial dysfunction on the proteome level, LC-MSE-based bottom-up, label-free differential proteomics expression analysis was used to monitor protein level changes in SH-SY5Y neuroblastoma cells induced by ETC-specific inhibitors (MPTP, 3-NP, sodium azide, antimycin A, and oligomycin). A total of 379 proteins were identified across the sample set and 75 of them were found to be differentially expressed (>30% fold change). Complex-specific inhibition of the five ETS complexes were expected to result in the aberrant regulation of different molecular pathways, but the bioinformatics analysis of the LC-MSMS data showed that the differentially expressed proteins were mostly involved in similar metabolic processes. The findings suggest that the complex-specific alterations may not be directly linked to neurodegenerative pathways, but could be considered contributors. Moreover, the proteins that showed the highest protein expression difference (>60% fold change) are involved in pathways regarding protein-folding and response to unfolded proteins. The results indicate that protein misfolding pathways might have a central role in the genesis and progression of neurodegenerative diseases and that label-free LC-MSMS proteomics analysis is an invaluable approach for studying of molecular pathways in neurodegeneration.
{"title":"Proteomics analysis of mitochondrial dysfunction triggered by complex specific electron transport chain inhibitors reveals common pathways involving protein misfolding in an SH-SY5Y in vitro cell model","authors":"B. Sahin, A. Baykal","doi":"10.3906/BIY-1702-44","DOIUrl":"https://doi.org/10.3906/BIY-1702-44","url":null,"abstract":"Mitochondrial dysfunction has been previously identified in neurodegenerative diseases such as Alzheimer disease, Huntington disease, and Parkinson disease. Chemical inhibition of the mitochondrial electron transport chain (ETC) was shown to trigger symptoms in animal models similar to those observed in human neurodegenerative diseases. In order to understand the effect of mitochondrial dysfunction on the proteome level, LC-MSE-based bottom-up, label-free differential proteomics expression analysis was used to monitor protein level changes in SH-SY5Y neuroblastoma cells induced by ETC-specific inhibitors (MPTP, 3-NP, sodium azide, antimycin A, and oligomycin). A total of 379 proteins were identified across the sample set and 75 of them were found to be differentially expressed (>30% fold change). Complex-specific inhibition of the five ETS complexes were expected to result in the aberrant regulation of different molecular pathways, but the bioinformatics analysis of the LC-MSMS data showed that the differentially expressed proteins were mostly involved in similar metabolic processes. The findings suggest that the complex-specific alterations may not be directly linked to neurodegenerative pathways, but could be considered contributors. Moreover, the proteins that showed the highest protein expression difference (>60% fold change) are involved in pathways regarding protein-folding and response to unfolded proteins. The results indicate that protein misfolding pathways might have a central role in the genesis and progression of neurodegenerative diseases and that label-free LC-MSMS proteomics analysis is an invaluable approach for studying of molecular pathways in neurodegeneration.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"765-784"},"PeriodicalIF":2.2,"publicationDate":"2017-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1702-44","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45905019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
D. Ćujić, M. Kosanović, M. J. Krivokuća, L. Vićovac, M. Janković
Galectins (gals) are s-galactoside binding lectins, involved in many processes at the fetomaternal interface where they can exert their roles both in and out of cells. The aim of this work was to explore the extracellular presence/release of HTR-8/SVneo extravillous trophoblast cell line galectins. To that end, conditioned medium (CM) from HTR-8/SVneo cell culture was fractionated into a high molecular mass fraction (HMF) and low molecular mass fraction (LMF) using 100 kDa cut-off concentrators. In addition, extracellular vesicles (EVs) were isolated from CM by ultracentrifugation. Size and shape of the EVs were analyzed by transmission electron microscopy and their galectin mRNA content was determined by real-time PCR. The presence of galectins in fractions of HTR-8/SVneo CM and EVs was detected by western blot. All three galectins expressed by HTR-8/SVneo cells (gal-1, gal-3, and gal-8) were detected in the CM, HMF, and EVs, while only gal-1 was found in the LMF. In addition, EVs contained all three galectin mRNAs. These results reveal that free, complexed, and EV-associated forms of galectins were released from extravillous trophoblast cells, suggesting their potential to exert extracellular functions both in their immediate vicinity at the fetomaternal interface and distant locations.
{"title":"Extracellular presence/release of galectins from HTR-8/SVneo extravillous trophoblast cells*","authors":"D. Ćujić, M. Kosanović, M. J. Krivokuća, L. Vićovac, M. Janković","doi":"10.3906/BIY-1704-11","DOIUrl":"https://doi.org/10.3906/BIY-1704-11","url":null,"abstract":"Galectins (gals) are s-galactoside binding lectins, involved in many processes at the fetomaternal interface where they can exert their roles both in and out of cells. The aim of this work was to explore the extracellular presence/release of HTR-8/SVneo extravillous trophoblast cell line galectins. To that end, conditioned medium (CM) from HTR-8/SVneo cell culture was fractionated into a high molecular mass fraction (HMF) and low molecular mass fraction (LMF) using 100 kDa cut-off concentrators. In addition, extracellular vesicles (EVs) were isolated from CM by ultracentrifugation. Size and shape of the EVs were analyzed by transmission electron microscopy and their galectin mRNA content was determined by real-time PCR. The presence of galectins in fractions of HTR-8/SVneo CM and EVs was detected by western blot. All three galectins expressed by HTR-8/SVneo cells (gal-1, gal-3, and gal-8) were detected in the CM, HMF, and EVs, while only gal-1 was found in the LMF. In addition, EVs contained all three galectin mRNAs. These results reveal that free, complexed, and EV-associated forms of galectins were released from extravillous trophoblast cells, suggesting their potential to exert extracellular functions both in their immediate vicinity at the fetomaternal interface and distant locations.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"843-848"},"PeriodicalIF":2.2,"publicationDate":"2017-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1704-11","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48104352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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