PeerJ Preprints: Cell Biologyhttps://peerj.com/preprints/index.atom?journal=peerj&subject=800Cell Biology articles published in PeerJ PreprintsKnockdown of AMP-activated protein kinase α2 impairs epithelial-mesenchymal transition in rat renal tubular epithelial cells by downregulating v-ets erythroblastosis virus E26 oncogene homolog-1 and ribosomal protein S6 kinase A1https://peerj.com/preprints/279922019-12-022019-12-02Xiaoming YinFujiang MaXu FanQi ZhaoXin LiuYi Yang
Background. Epithelial mesenchymal transition (EMT) plays an important regulatory role in obstructive nephropathy and renal fibrosis. As an intracellular energy sensor, AMP-activated protein kinase (AMPK) is essential in the process of EMT. The aim of this study was to reveal changes in the expression of AMPKα2 and to elucidate which AMPKα2 genes play a role during EMT. Methods. In this study, TGF-β1 was used to induce EMT in normal rat renal tubular epithelial (NRK-52E) cells. The shAMPKα2 lentivirus was used to interfere with AMPKα2 expression in EMT-derived NRK-52E cells, where AMPKα2 expression and EMT were detected. Differential gene expression after the AMPKα2 knockdown in EMT-derived NRK-52E cells was examined using a gene microarray. Possible regulatory pathways were analyzed using ingenuity pathway analysis (IPA) and differentially expressed genes were partially verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Results. It was found that AMPKα2 was upregulated in TGF-β1-induced EMT-derived NRK-52E cells. EMT progression was significantly inhibited after the expression of AMPKα2 was downregulated by the shAMPKα2 lentivirus. A total of 1,588 differentially expressed genes were detected after the AMPKα2 knockdown in NRK-52E cells in which EMT occurred. The ERK/MAPK pathway was significantly impaired after the AMPKα2 knockdown, as indicated by the IPA analysis. Furthermore, qRT-PCR and western blot results revealed that the expression of AMPKα2, v-ets erythroblastosis virus E26 oncogene homolog-1 (ETS1), and ribosomal protein S6 kinase A1 (RPS6KA1) was upregulated after EMT in NRK-52E cells, while expression of ETS1 and RPS6KA1 was downregulated after the AMPKα2 knockdown. Conclusions. AMPKα2 plays an important role in the regulation of rat renal tubular EMT, which may be achieved by modulating ETS1 and RPS6KA1 in the ERK/MAPK pathway.
Background. Epithelial mesenchymal transition (EMT) plays an important regulatory role in obstructive nephropathy and renal fibrosis. As an intracellular energy sensor, AMP-activated protein kinase (AMPK) is essential in the process of EMT. The aim of this study was to reveal changes in the expression of AMPKα2 and to elucidate which AMPKα2 genes play a role during EMT. Methods. In this study, TGF-β1 was used to induce EMT in normal rat renal tubular epithelial (NRK-52E) cells. The shAMPKα2 lentivirus was used to interfere with AMPKα2 expression in EMT-derived NRK-52E cells, where AMPKα2 expression and EMT were detected. Differential gene expression after the AMPKα2 knockdown in EMT-derived NRK-52E cells was examined using a gene microarray. Possible regulatory pathways were analyzed using ingenuity pathway analysis (IPA) and differentially expressed genes were partially verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Results. It was found that AMPKα2 was upregulated in TGF-β1-induced EMT-derived NRK-52E cells. EMT progression was significantly inhibited after the expression of AMPKα2 was downregulated by the shAMPKα2 lentivirus. A total of 1,588 differentially expressed genes were detected after the AMPKα2 knockdown in NRK-52E cells in which EMT occurred. The ERK/MAPK pathway was significantly impaired after the AMPKα2 knockdown, as indicated by the IPA analysis. Furthermore, qRT-PCR and western blot results revealed that the expression of AMPKα2, v-ets erythroblastosis virus E26 oncogene homolog-1 (ETS1), and ribosomal protein S6 kinase A1 (RPS6KA1) was upregulated after EMT in NRK-52E cells, while expression of ETS1 and RPS6KA1 was downregulated after the AMPKα2 knockdown. Conclusions. AMPKα2 plays an important role in the regulation of rat renal tubular EMT, which may be achieved by modulating ETS1 and RPS6KA1 in the ERK/MAPK pathway.A lipid-invasion model for Alzheimer’s Diseasehttps://peerj.com/preprints/278112019-11-182019-11-18Jonathan D Rudge
This paper describes a potential new explanation for Alzheimer’s disease (AD), referred to here as the lipid-invasion model. It proposes that AD is primarily caused by the influx of lipids following the breakdown of the blood brain barrier (BBB). The model argues that a principal role of the BBB is to protect the brain from external lipid access. When the BBB is damaged, it allows a mass influx of (mainly albumin-bound) free fatty acids (FFAs) and lipid-rich lipoproteins to the brain, which in turn causes neurodegeneration, amyloidosis, tau tangles and other AD characteristics. The model also argues that, whilst β-amyloid causes neurodegeneration, as is widely argued, its principal role in the disease lies in damaging the BBB. It is the external lipids, entering as a consequence, that are the primary drivers of neurodegeneration in AD., especially FFAs, which induce oxidative stress, stimulate microglia-driven neuroinflammation, and inhibit neurogenesis. Simultaneously, the larger, more lipid-laden lipoproteins, characteristic of the external plasma but not the CNS, cause endosomal-lysosomal abnormalities, amyloidosis and the formation of tau tangles, all characteristic of AD. In most cases (certainly in late-onset, noninherited forms of the disease) amyloidosis and tau tangle formation are consequences of this external lipid invasion, and in many ways more symptomatic of the disease than causative. In support of this, it is argued that the pattern of damage caused by the influx of FFAs into the brain is likely to resemble the neurodegeneration seen in alcohol-related brain damage (ARBD), a disease that shows many similarities to AD, including the areas of the brain it affects. The fact that neurodegeneration is far more pronounced in AD than in ARBD, and characterised by other features, such as amyloidosis and tau tangles, most likely results from the greater heterogeneity of the lipid assault in AD compared with ethanol alone. The lipid-invasion model, described here, arguably provides the first cohesive, multi-factorial explanation of AD that accounts for all currently known major risk factors, and explains all AD-associated pathologies, including those, such as endosomal-lysosomal dysfunction and excessive lipid droplet formation, that are not well-accounted for in other explanation of this disease.
This paper describes a potential new explanation for Alzheimer’s disease (AD), referred to here as the lipid-invasion model. It proposes that AD is primarily caused by the influx of lipids following the breakdown of the blood brain barrier (BBB). The model argues that a principal role of the BBB is to protect the brain from external lipid access. When the BBB is damaged, it allows a mass influx of (mainly albumin-bound) free fatty acids (FFAs) and lipid-rich lipoproteins to the brain, which in turn causes neurodegeneration, amyloidosis, tau tangles and other AD characteristics. The model also argues that, whilst β-amyloid causes neurodegeneration, as is widely argued, its principal role in the disease lies in damaging the BBB. It is the external lipids, entering as a consequence, that are the primary drivers of neurodegeneration in AD., especially FFAs, which induce oxidative stress, stimulate microglia-driven neuroinflammation, and inhibit neurogenesis. Simultaneously, the larger, more lipid-laden lipoproteins, characteristic of the external plasma but not the CNS, cause endosomal-lysosomal abnormalities, amyloidosis and the formation of tau tangles, all characteristic of AD. In most cases (certainly in late-onset, noninherited forms of the disease) amyloidosis and tau tangle formation are consequences of this external lipid invasion, and in many ways more symptomatic of the disease than causative. In support of this, it is argued that the pattern of damage caused by the influx of FFAs into the brain is likely to resemble the neurodegeneration seen in alcohol-related brain damage (ARBD), a disease that shows many similarities to AD, including the areas of the brain it affects. The fact that neurodegeneration is far more pronounced in AD than in ARBD, and characterised by other features, such as amyloidosis and tau tangles, most likely results from the greater heterogeneity of the lipid assault in AD compared with ethanol alone. The lipid-invasion model, described here, arguably provides the first cohesive, multi-factorial explanation of AD that accounts for all currently known major risk factors, and explains all AD-associated pathologies, including those, such as endosomal-lysosomal dysfunction and excessive lipid droplet formation, that are not well-accounted for in other explanation of this disease.CYTO Lab Hacks: A platform for the exchange of innovations in cytometryhttps://peerj.com/preprints/279442019-10-222019-10-22Cláudia BispoBunny CotleurChristopher HallVirginia LitwinJakub NedbalBetsy Ohlsson-Wilhelm
This article reports on a conference workshop conducted at CYTO 2019. This workshop centered on an online directory for non-commercial cytometry innovations called CYTO Lab Hacks. The CYTO Lab Hacks website is being developed to become a curated platform to collate and to promote cytometry related materials developed by the wider scientific community. The website will present brief summaries and links to repositories with experimental protocols, descriptions of hardware changes, document templates, software code, and other innovations. The workshop outcomes, summarized in this manuscript, cover the topics of the website functionality and user experience, organization of the volunteer task force, and understanding the needs of the cytometry community in respect to sharing innovations.
This article reports on a conference workshop conducted at CYTO 2019. This workshop centered on an online directory for non-commercial cytometry innovations called CYTO Lab Hacks. The CYTO Lab Hacks website is being developed to become a curated platform to collate and to promote cytometry related materials developed by the wider scientific community. The website will present brief summaries and links to repositories with experimental protocols, descriptions of hardware changes, document templates, software code, and other innovations. The workshop outcomes, summarized in this manuscript, cover the topics of the website functionality and user experience, organization of the volunteer task force, and understanding the needs of the cytometry community in respect to sharing innovations.Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cell derived from human follicular fluid to oocyte like cellhttps://peerj.com/preprints/280062019-10-072019-10-07Mahin Taheri MoghadamAli Reza Eftekhari MoghadamGhasem SakiRoshan Nikbakht
Background. To study the effect of Bone morphogenetic protein 15 on differentiation potential of mesenchymal stem cell derived from human follicular fluid to oocyte like cell. Methods. Human FF derived cells were collected from 78 women in assisted fertilization program, and cultured in differentiation medium containing human recombinant BMP15 for 21 days. Mesenchymal stem cells and OLCs were characterized by real-time PCR and immunocytochemistry (ICC) staining. Results. MSCs expressed germ line stem cell markers, such as OCT4 and NANOG. After 15 days, OLCs formed and expressed zona pellucida markers (ZP2, ZP3), and reached 20 – 30 µm in diameters. Ten days after induction with BMP15, round cells remarkably developed, and the maximum size of OLCs reached 115 µm. Finally, a decrease ranging from 0.04 to 4.5 in the expression of pluripotency and oocyte specific markers was observed in the cells cultured in BMP15 supplemented medium. Our work demonstrates, FF derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in stimulating the differentiation of these cells, which may give an in vitro model to examine human germ cell development.
Background. To study the effect of Bone morphogenetic protein 15 on differentiation potential of mesenchymal stem cell derived from human follicular fluid to oocyte like cell. Methods. Human FF derived cells were collected from 78 women in assisted fertilization program, and cultured in differentiation medium containing human recombinant BMP15 for 21 days. Mesenchymal stem cells and OLCs were characterized by real-time PCR and immunocytochemistry (ICC) staining. Results. MSCs expressed germ line stem cell markers, such as OCT4 and NANOG. After 15 days, OLCs formed and expressed zona pellucida markers (ZP2, ZP3), and reached 20 – 30 µm in diameters. Ten days after induction with BMP15, round cells remarkably developed, and the maximum size of OLCs reached 115 µm. Finally, a decrease ranging from 0.04 to 4.5 in the expression of pluripotency and oocyte specific markers was observed in the cells cultured in BMP15 supplemented medium. Our work demonstrates, FF derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in stimulating the differentiation of these cells, which may give an in vitro model to examine human germ cell development.Multifaceted role of tensins in cancerhttps://peerj.com/preprints/279902019-09-272019-09-27Abdulaziz AlfahedTeresa P RaposoMohammad Ilyas
Tensins are structural adaptor proteins localized at focal adhesions. Tensins can act as mechanosensors and participate in the transduction of biochemical signals from the extracellular matrix to the cytoskeleton, acting as an interface able to alter cell behavior in responses to changes in their surrounding environment. This review aims to provide a concise summary of the main functions of the four known tensins in cell and cancer biology, their homology and recently unveiled signaling mechanisms. We focus specifically on how tensin 4 (TNS4/Cten) may contribute to cancer both as an oncogene supporting metastasis and as tumour suppressor in different types of tissue. A better understanding of the cancer mechanistics involving tensins may provide the rationale for development of specific therapeutic strategies.
Tensins are structural adaptor proteins localized at focal adhesions. Tensins can act as mechanosensors and participate in the transduction of biochemical signals from the extracellular matrix to the cytoskeleton, acting as an interface able to alter cell behavior in responses to changes in their surrounding environment. This review aims to provide a concise summary of the main functions of the four known tensins in cell and cancer biology, their homology and recently unveiled signaling mechanisms. We focus specifically on how tensin 4 (TNS4/Cten) may contribute to cancer both as an oncogene supporting metastasis and as tumour suppressor in different types of tissue. A better understanding of the cancer mechanistics involving tensins may provide the rationale for development of specific therapeutic strategies.Cell lineage trees: the central structure plus key dynamics of biological aging and formulating the limiting problem of comprehensive organismal rejuvenationhttps://peerj.com/preprints/278212019-09-192019-09-19Attila Csordas
The argument makes the case for cell lineage trees and cell tree dynamics to be considered as the central structure and process of understanding organismal level, multicellular biological aging. The limiting challenge of counteracting biological aging is comprehensive organismal rejuvenation. The central theoretical problem of comprehensive biological rejuvenation is to find an algorithm to restore the balance and maintain the healthy dynamics of the aging organismal cell lineage tree. The most comprehensive medical solution of biological aging needs to use individual cell lineage trees as a central tool for diagnosis and treatment.
The argument makes the case for cell lineage trees and cell tree dynamics to be considered as the central structure and process of understanding organismal level, multicellular biological aging. The limiting challenge of counteracting biological aging is comprehensive organismal rejuvenation. The central theoretical problem of comprehensive biological rejuvenation is to find an algorithm to restore the balance and maintain the healthy dynamics of the aging organismal cell lineage tree. The most comprehensive medical solution of biological aging needs to use individual cell lineage trees as a central tool for diagnosis and treatment.Proximate composition and quantitative analysis of benzoyl peroxide and benzoic acid in the wheat flour samples: wheat flour qualityhttps://peerj.com/preprints/279692019-09-182019-09-18Numrah NisarFaiza MustafaArifa TahiraRashad Waseem Khan QadriYaodong YangMuhammad Imran Khan
Background. Extensive milling processes have deprived wheat flour from essential nutrients. Objective of the current study was to assess the nutritive quality of commercial wheat flour (soft flour/SF) through analyses of proximate composition and functional properties as well as quantification of benzoyl peroxide (BP; added as bleaching agent in the SF). Methods. Test samples included commercial soft flour samples purchased from the local supplier from different flour mills (with additives) and a control sample without additives was prepared by grinding the seeds harvested from wheat crop grown in the experimental field of University of Agriculture, Faisalabad, under optimized field conditions without any fertilizer and insecticide. Benzoyl peroxide and Benzoic Acid quantification was performed through High Performance Liquid Chromatography Results. Results when compared with the whole wheat flour (WF; never received additives) indicated that SF had lesser fiber, protein and ash contents, whereas, higher damaged starch, fat, gluten and bulk density. A parallel experiment under selected conditions (temperature, time and solute concentration) showed dissociation of BP into BA soon after the exposure. Observed BA range (13.77 mg/g after 16hrs) in SF and exposure level assessment (44.3±1.36 mg/kg/BW) showed higher intake of BA on the consumption of SF. Results revealed superiority of WF over SF in nutritive qualities as well as free of toxicants such as BA. KEYWORDS: Benzoyl peroxide; Benzoic acid; Soft Flour; Whole Wheat Flour; High Performance Liquid Chromatography
Background. Extensive milling processes have deprived wheat flour from essential nutrients. Objective of the current study was to assess the nutritive quality of commercial wheat flour (soft flour/SF) through analyses of proximate composition and functional properties as well as quantification of benzoyl peroxide (BP; added as bleaching agent in the SF). Methods. Test samples included commercial soft flour samples purchased from the local supplier from different flour mills (with additives) and a control sample without additives was prepared by grinding the seeds harvested from wheat crop grown in the experimental field of University of Agriculture, Faisalabad, under optimized field conditions without any fertilizer and insecticide. Benzoyl peroxide and Benzoic Acid quantification was performed through High Performance Liquid Chromatography Results. Results when compared with the whole wheat flour (WF; never received additives) indicated that SF had lesser fiber, protein and ash contents, whereas, higher damaged starch, fat, gluten and bulk density. A parallel experiment under selected conditions (temperature, time and solute concentration) showed dissociation of BP into BA soon after the exposure. Observed BA range (13.77 mg/g after 16hrs) in SF and exposure level assessment (44.3±1.36 mg/kg/BW) showed higher intake of BA on the consumption of SF. Results revealed superiority of WF over SF in nutritive qualities as well as free of toxicants such as BA. KEYWORDS: Benzoyl peroxide; Benzoic acid; Soft Flour; Whole Wheat Flour; High Performance Liquid ChromatographyLive cell imaging of nuclear actin filaments and heterochromatic repair foci in Drosophila and mouse cellshttps://peerj.com/preprints/279002019-08-142019-08-14Colby SeeDeepak AryaEmily LinIrene Chiolo
Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.
Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.Tubular membranes extended between monolayer cells, from solid spheroids, and from clustered hollow spheroids in Ishikawa endometrial cell cultures can carry chromatin granules and mitonucleonshttps://peerj.com/preprints/278952019-08-122019-08-12Honoree Fleming
Membrane tubular extensions, recently recognized an important communication element in mammalian cells are demonstrated to form in Ishikawa endometrial epithelial cells growing in monolayers, and to extend from solid spheroids and from clustered hollow spheroids. Two kinds of chromatin cargoes travel through these tubules. Chromatin granules can pass through an endometrial tubule bridge extending from one monolayer fragment to another. The passage of granules over time from one of the fragments appears to support the self-assembly of nuclei in the other colony fragment. Similarly, in a process detected by observing an open-ended membrane tubule extending from a solid cell spheroid, a nucleus was observed to form over a period of 3 hours. Indications are that chromatin granules such as those observed in the amitotic processes of epithelial dome cell formation and of hollow spheroid cell formation are contributing to the build up of nuclei. Mitonucleons, a transient subcellular organelle consisting of fused mitochondria intimately associated with aggregated chromatin are also observed to pass through tubular membrane extensions. Multiple membrane extensions can be shown to to extend from clusters of unicellular polyploid hollow spheroids whose formation is described for the first time in this paper.
Membrane tubular extensions, recently recognized an important communication element in mammalian cells are demonstrated to form in Ishikawa endometrial epithelial cells growing in monolayers, and to extend from solid spheroids and from clustered hollow spheroids. Two kinds of chromatin cargoes travel through these tubules. Chromatin granules can pass through an endometrial tubule bridge extending from one monolayer fragment to another. The passage of granules over time from one of the fragments appears to support the self-assembly of nuclei in the other colony fragment. Similarly, in a process detected by observing an open-ended membrane tubule extending from a solid cell spheroid, a nucleus was observed to form over a period of 3 hours. Indications are that chromatin granules such as those observed in the amitotic processes of epithelial dome cell formation and of hollow spheroid cell formation are contributing to the build up of nuclei. Mitonucleons, a transient subcellular organelle consisting of fused mitochondria intimately associated with aggregated chromatin are also observed to pass through tubular membrane extensions. Multiple membrane extensions can be shown to to extend from clusters of unicellular polyploid hollow spheroids whose formation is described for the first time in this paper.Internal sensation of pleasure can be explained as a specific conformation of semblance: Inference from electrophysiological findingshttps://peerj.com/preprints/278862019-08-062019-08-06Kunjumon Vadakkan
Semblance hypothesis was able to find a solution for the generation of first- person internal sensation of memory along with provisions for behavioral motor actions. The derived inter-postsynaptic functional LINK (IPL) mechanism was able to explain a large number of findings from different levels of the system ranging from perception to sleep. It was possible to explain long-term potentiation (LTP) as the effect of experimental scaling-up of the changes occurring during natural learning. By keeping the latter relationship as a baseline, it was possible to explain long-term depression (LTD) observed in the nucleus accumbens (NAc), a scaled-up change of a mechanism responsible for inducing internal sensation of pleasure. This mechanism provides inter-connectable explanations for the attenuation of postsynaptic potentials, reduced ring of medium spiny neurons and the finding that LTD induced by stimulation of one pathway to NAc occludes the LTD induction by another pathway.
Semblance hypothesis was able to find a solution for the generation of first- person internal sensation of memory along with provisions for behavioral motor actions. The derived inter-postsynaptic functional LINK (IPL) mechanism was able to explain a large number of findings from different levels of the system ranging from perception to sleep. It was possible to explain long-term potentiation (LTP) as the effect of experimental scaling-up of the changes occurring during natural learning. By keeping the latter relationship as a baseline, it was possible to explain long-term depression (LTD) observed in the nucleus accumbens (NAc), a scaled-up change of a mechanism responsible for inducing internal sensation of pleasure. This mechanism provides inter-connectable explanations for the attenuation of postsynaptic potentials, reduced ring of medium spiny neurons and the finding that LTD induced by stimulation of one pathway to NAc occludes the LTD induction by another pathway.