PeerJ Preprints: Molecular Biologyhttps://peerj.com/preprints/index.atom?journal=peerj&subject=2100Molecular Biology articles published in PeerJ PreprintsA 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.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.Using environmental DNA to monitor the reintroduction success of the Rhine sculpin (Cottus rhenanus) in a restored streamhttps://peerj.com/preprints/275742019-09-272019-09-27Christopher A HempelBianca PeinertArne J BeermannVasco ElbrechtJan-Niklas MacherTill-Hendrik MacherGunnar JacobsFlorian Leese
Freshwaters face some of the highest rates of species loss, caused by strong human impact. To decrease this strong impact, ecological restorations are increasingly applied to restore and maintain the natural ecological status of freshwaters. Their ecological status can be determined by assessing the presence of indicator species (e.g. certain fish species), which is called biomonitoring. However, traditional biomonitoring of fish, such as electrofishing, is often challenging and invasive. To augment traditional biomonitoring of fish, the analysis of environmental DNA (eDNA) has recently been proposed as an alternative, sensitive approach. The present study employed this modern approach to monitor the Rhine sculpin (Cottus rhenanus), a fish species that has been reintroduced into a recently restored stream within the Emscher catchment in Germany, in order to validate the success of the applied restorations and to monitor the species’ dispersal. We monitored the dispersal of the Rhine sculpin using replicated 12S end-point PCR eDNA surveillance at a fine spatial and temporal scale. In that way, we investigated if eDNA analysis can be applied for freshwater assessments. We also performed traditional electrofishing in one instance to validate our eDNA-based approach. We could track the dispersal of the Rhine sculpin and showed a higher dispersal potential of the species than we assumed. We validated the species’ dispersal across a potential dispersal barrier via eDNA detection and showed a steep increase of positive detections once the reintroduced population had established. In contrast to that, false negative eDNA results occurred at early reintroduction stages. Our results show that eDNA detection can be used to confirm and monitor reintroductions and to contribute to the assessment and modelling of ecological status of streams.
Freshwaters face some of the highest rates of species loss, caused by strong human impact. To decrease this strong impact, ecological restorations are increasingly applied to restore and maintain the natural ecological status of freshwaters. Their ecological status can be determined by assessing the presence of indicator species (e.g. certain fish species), which is called biomonitoring. However, traditional biomonitoring of fish, such as electrofishing, is often challenging and invasive. To augment traditional biomonitoring of fish, the analysis of environmental DNA (eDNA) has recently been proposed as an alternative, sensitive approach. The present study employed this modern approach to monitor the Rhine sculpin (Cottus rhenanus), a fish species that has been reintroduced into a recently restored stream within the Emscher catchment in Germany, in order to validate the success of the applied restorations and to monitor the species’ dispersal. We monitored the dispersal of the Rhine sculpin using replicated 12S end-point PCR eDNA surveillance at a fine spatial and temporal scale. In that way, we investigated if eDNA analysis can be applied for freshwater assessments. We also performed traditional electrofishing in one instance to validate our eDNA-based approach. We could track the dispersal of the Rhine sculpin and showed a higher dispersal potential of the species than we assumed. We validated the species’ dispersal across a potential dispersal barrier via eDNA detection and showed a steep increase of positive detections once the reintroduced population had established. In contrast to that, false negative eDNA results occurred at early reintroduction stages. Our results show that eDNA detection can be used to confirm and monitor reintroductions and to contribute to the assessment and modelling of ecological status of streams.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.Functional characterization of a new maize heat shock transcription factor gene ZmHsf01 playing important roles in thermotolerancehttps://peerj.com/preprints/279872019-09-262019-09-26Huaning ZhangGuoliang LiYuanyuan ZhangYujie ZhangHongbo ShaoDong HuXiulin Guo
Background. The yield of maize crop is influenced seriously by heat waves. Plant heat shock transcription factors (Hsfs) play a key regulatory role in heat shock signal transduction pathway. Method. In this study, a new heat shock transcription factor gene, ZmHsf01 (accession number: MK888854) , was cloned from maize young leaves using homologous cloning method. The transcriptional level of ZmHsf01 were detected by qRT-PCR in different tissues or under heat shock, abscisic acid (ABA) and hydrogen peroxide (H2O2) treatment. The transgenic yeast and Arabidopsis were used to study the gene function of ZmHsf01. Result. The coding sequence (CDS) of ZmHsf01 was 1176 bp and encoded a protein that consisted of 391 amino acids. The homologous analysis result showed that ZmHsf01 and SbHsfA2d had the highest protein sequence identity. Subcellular localization experiments demonstrated that ZmHsf01 is localized to the nucleus. ZmHsf01 was expressed in many maize tissues and was up-regulated by heat stress. ZmHsf01 was up-regulated in roots and down-regulated in leaves by ABA and H2O2treatments. In yeast, ZmHsf01-overexpressing cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 complemented the thermotolerance defects of athsfa2 mutant and ZmHsf01-overexpressing lines presented enhanced basal and acquired thermotolerance. Compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed increased chlorophyll content after heat stress. The expression level of heat shock protein genes was up-regulated higher in ZmHsf01-overexpressing Arabidopsis seedlings than that in WT. These results suggested that ZmHsf01 plays a vital role in plant response to heat stress.
Background. The yield of maize crop is influenced seriously by heat waves. Plant heat shock transcription factors (Hsfs) play a key regulatory role in heat shock signal transduction pathway. Method. In this study, a new heat shock transcription factor gene, ZmHsf01 (accession number: MK888854), was cloned from maize young leaves using homologous cloning method. The transcriptional level of ZmHsf01 were detected by qRT-PCR in different tissues or under heat shock, abscisic acid (ABA) and hydrogen peroxide (H2O2) treatment. The transgenic yeast and Arabidopsis were used to study the gene function of ZmHsf01. Result. The coding sequence (CDS) of ZmHsf01 was 1176 bp and encoded a protein that consisted of 391 amino acids. The homologous analysis result showed that ZmHsf01 and SbHsfA2dhad the highest protein sequence identity. Subcellular localization experiments demonstrated that ZmHsf01 is localized to the nucleus. ZmHsf01 was expressed in many maize tissues and was up-regulated by heat stress. ZmHsf01 was up-regulated in roots and down-regulated in leaves by ABA and H2O2treatments. In yeast, ZmHsf01-overexpressing cells showed increased thermotolerance. In Arabidopsis seedlings, ZmHsf01 complemented the thermotolerance defects of athsfa2 mutant and ZmHsf01-overexpressing lines presented enhanced basal and acquired thermotolerance. Compared to wild type (WT) seedlings, ZmHsf01-overexpressing lines showed increased chlorophyll content after heat stress. The expression level of heat shock protein genes was up-regulated higher in ZmHsf01-overexpressing Arabidopsis seedlings than that in WT. These results suggested that ZmHsf01 plays a vital role in plant response to heat stress.Antimicrobial resistance and genetic relationship among enterococci from siblings and non-siblings Heliconius erato phyllis caterpillarshttps://peerj.com/preprints/279762019-09-232019-09-23Rosana HuffRebeca Inhoque PereiraCaroline PissettiAldo Mellender de AraújoPedro Alves d’AzevedoJeverson FrazzonAna Paula Guedes Frazzon
Background: Studies evaluating bacterial in insects could provide information about host-microorganism-environment interactions. The gut community is recognized to have a profound effect on various physiological functions of the insects. Enterococcus is part of the gut community in humans andanimals, as well as in the guts of insects. The presence and antimicrobialresistance profileof enterococci are well studied in different animals; however, data in Heliconius erato phyllis (Lepidoptera; Nymphalidae), do not yet exist. Therefore, the aims of this study were to evaluate species distribution, antimicrobial resistance profile, virulence genes and genetic relationship amongenterococci isolated from fecal samples of siblings and non-siblings H. erato phyllis caterpillars collected from different places in South Brazil.
Methods: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n=12) were collected from fifth instar caterpillars, inoculated in selective medium and fifteen colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, submitted to antimicrobial susceptibility tests by disk diffusion method, and screened for resistance and virulence genes by PCR. The genetic relationships among the strains were determined using pulsed-field gel electrophoresis (PFGE).
Results: A total of 178 enterococci were identified as: Enterococcus casseliflavus (74.15%; n=132),E. mundtii (21.34%; n=38), E. faecalis (1.12%; n=2) and Enterococcus sp. (3.37%; n=6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed. One hundred and twenty (67.41%) out of the 178 isolates showed resistance to at least one compound and 6 (3.37%) were multidrug-resistant.None of the erythromycin-resistant strains was positive to erm(B) and msrC genes. The virulence genes esp, ace and gelE were observed in 35%, 7% and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, being four patterns composed by strains from sibling caterpillars.
Conclusion: Enterococcus casseliflavus was the dominant species in fecal samples of fifth instar caterpillars. Resistant enterococci strains could be related to environmental pollution or linked to environmental resistome. The PFGE analysis showed related genetic relationship among some strains, suggesting that the enterococci isolated from fecal samples of fifth instar sibling caterpillars might have come from common sources, by diet (herbivory) and/or via vertical transmission (through egg surface). Further studies will be conducted to better understand the role of Enterococcus on the microbial gastrointestinal tract community of these insects, and the mechanisms involved in acquisition and maintenance of these bacteria.
Background: Studies evaluating bacterial in insects could provide information about host-microorganism-environment interactions. The gut community is recognized to have a profound effect on various physiological functions of the insects. Enterococcus is part of the gut community in humans andanimals, as well as in the guts of insects. The presence and antimicrobialresistance profileof enterococci are well studied in different animals; however, data in Heliconius erato phyllis (Lepidoptera; Nymphalidae), do not yet exist. Therefore, the aims of this study were to evaluate species distribution, antimicrobial resistance profile, virulence genes and genetic relationship amongenterococci isolated from fecal samples of siblings and non-siblings H. erato phyllis caterpillars collected from different places in South Brazil.Methods: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n=12) were collected from fifth instar caterpillars, inoculated in selective medium and fifteen colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, submittedto antimicrobial susceptibility tests by disk diffusion method, and screened for resistance and virulencegenes by PCR. The genetic relationships among the strains were determined using pulsed-field gel electrophoresis (PFGE).Results: A total of 178 enterococci were identified as: Enterococcus casseliflavus (74.15%; n=132),E. mundtii (21.34%; n=38), E. faecalis (1.12%; n=2) and Enterococcus sp. (3.37%; n=6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed. One hundred and twenty (67.41%) out of the 178 isolates showed resistance to at least one compound and 6 (3.37%) were multidrug-resistant.None of the erythromycin-resistant strains was positive to erm(B) and msrC genes. The virulence genes esp, ace and gelE were observed in 35%, 7% and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, being four patterns composed by strains from sibling caterpillars.Conclusion: Enterococcus casseliflavus was the dominant species in fecal samples of fifth instarcaterpillars. Resistant enterococci strains could be related to environmental pollution or linked to environmental resistome. The PFGE analysis showed related genetic relationship among some strains, suggesting that the enterococci isolated from fecal samples of fifth instar sibling caterpillars might have come from common sources, by diet (herbivory) and/or via vertical transmission (through egg surface). Further studies will be conducted to better understand the role of Enterococcus on the microbial gastrointestinal tract community of these insects, and the mechanisms involved in acquisition and maintenance of these bacteria.A guide to carrying out a phylogenomic target sequence capture projecthttps://peerj.com/preprints/279682019-09-182019-09-18Tobias AndermannMaria Fernanda Torres JimenezPável Matos-MaravíRomina BatistaJosé L Blanco-PastorA. Lovisa S GustafssonLogan KistlerIsabel M LiberalBengt OxelmanChristine D BaconAlexandre Antonelli
High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing efforts on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing sequencing depth. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth and has proven to produce powerful, large multi-locus DNA sequence datasets of selected loci, suitable for phylogenetic analyses. However, target capture requires careful theoretical and practical considerations, which will greatly affect the success of the experiment. Here we provide an easy-to-follow flowchart for adequately designing phylogenomic target capture experiments, and we discuss necessary considerations and decisions from the first steps in the lab to the final bioinformatic processing of the sequence data. We particularly discuss issues and challenges related to the taxonomic scope, sample quality, and available genomic resources of target capture projects and how these issues affect all steps from bait design to the bioinformatic processing of the data. Altogether this review outlines a roadmap for future target capture experiments and is intended to assist researchers with making informed decisions for designing and carrying out successful phylogenetic target capture studies
High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing efforts on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing sequencing depth. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth and has proven to produce powerful, large multi-locus DNA sequence datasets of selected loci, suitable for phylogenetic analyses. However, target capture requires careful theoretical and practical considerations, which will greatly affect the success of the experiment. Here we provide an easy-to-follow flowchart for adequately designing phylogenomic target capture experiments, and we discuss necessary considerations and decisions from the first steps in the lab to the final bioinformatic processing of the sequence data. We particularly discuss issues and challenges related to the taxonomic scope, sample quality, and available genomic resources of target capture projects and how these issues affect all steps from bait design to the bioinformatic processing of the data. Altogether this review outlines a roadmap for future target capture experiments and is intended to assist researchers with making informed decisions for designing and carrying out successful phylogenetic target capture studiesAn analysis of the mechanism of aging: endogenous viral stimulus and the deleterious effect of chronic inflammationhttps://peerj.com/preprints/274412019-09-132019-09-13Chingis Ochirov
This article describes a putative mechanism of aging based on the interaction of endogenous viral particles with the receptors of the innate immune system leading to producing pro-inflammatory cytokines. The innate immune response induces a complex of signaling pathways leading to senescence or tumorigenesis. The fate of a cell is depended on the activity of the p53 tumor-suppressive signaling pathway. Chronic inflammation is characterized by upregulation of the NF-kB signaling. The NF-kB protein stimulates the expression of matrix metalloproteinases (MMPs) leading to remodeling of extracellular matrix. The extracellular matrix alterations induce the loss of stem cell environment and their depletion. The innate immune system also mediates the PI3K-Akt-mTOR signaling pathway that inhibits autophagy and transforms energy metabolism providing cell senescence, high level of blood glucose, high lipid synthesis and mitochondrial alterations. The STAT3-HIF1 signaling pathway suppresses oxidative phosphorylation increasing ROS production and promoting the MAPK pathway leading to excessive cell proliferation. The increased ROS production causes the global DNA and histone demethylation contributing to retrotransposon reactivation whose activity leads to genome instability. However, the activity of retrotransposons may be partly explained by their role in adaptation. Among retrotransposons, endogenous retroviruses may be considered as an intrinsic stimulus for the innate immune system and are also able to avoid the adaptive immune system. Therefore, I consider endogenous retroviruses as promising targets in anti-aging therapies
This article describes a putative mechanism of aging based on the interaction of endogenous viral particles with the receptors of the innate immune system leading to producing pro-inflammatory cytokines. The innate immune response induces a complex of signaling pathways leading to senescence or tumorigenesis. The fate of a cell is depended on the activity of the p53 tumor-suppressive signaling pathway. Chronic inflammation is characterized by upregulation of the NF-kB signaling. The NF-kB protein stimulates the expression of matrix metalloproteinases (MMPs) leading to remodeling of extracellular matrix. The extracellular matrix alterations induce the loss of stem cell environment and their depletion. The innate immune system also mediates the PI3K-Akt-mTOR signaling pathway that inhibits autophagy and transforms energy metabolism providing cell senescence, high level of blood glucose, high lipid synthesis and mitochondrial alterations. The STAT3-HIF1 signaling pathway suppresses oxidative phosphorylation increasing ROS production and promoting the MAPK pathway leading to excessive cell proliferation. The increased ROS production causes the global DNA and histone demethylation contributing to retrotransposon reactivation whose activity leads to genome instability. However, the activity of retrotransposons may be partly explained by their role in adaptation. Among retrotransposons, endogenous retroviruses may be considered as an intrinsic stimulus for the innate immune system and are also able to avoid the adaptive immune system. Therefore, I consider endogenous retroviruses as promising targets in anti-aging therapiesThe detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assayhttps://peerj.com/preprints/279252019-08-292019-08-29Kai AokiTakehito SugasawaKouki YanazawaKoichi WatanabeTohru TakemasaYoshinori TakeuchiYuichi AitaNaoya YahagiYasuko YoshidaTomoaki KujiNanami SekineKaoru TakeuchiHaruna UedaYasushi KawakamiKazuhiro Takekoshi
BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.
BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.Candidate genes in coffee (Coffea arabica L.) leaves associated with rust (Hemileia vastatrix Berk. & Br) stresshttps://peerj.com/preprints/279232019-08-282019-08-28Fabián Echeverría-BeiruteSeth C. MurrayBenoit BertrandPatricia E. Klein
Background. Coffee leaf rust (CLR) caused by Hemileia vastatrix Berk. & Br, is one of the most threatening diseases for Coffea arabica L. It is hypothesized that host tolerance to CLR relies on non-race-specific resistance genes.
Methods. This study evaluated gene expression in leaves of two susceptible coffee cultivars (one inbred and one F1 hybrid) under different stress conditions: rust control (fungicide and untreated) and fruit thinning (thinned and un-thinned) treatments. RNA-seq analysis focused on the association of differentially expressed genes (DEGs) with CLR and associated the effect of the most significant genes into the phenotype, using regression and prediction statistical models.
Results. Gene expression and gene ontology (GO) analysis allowed identification of 100 genes associated with quantitative traits. From these, 88 were correlated with rust incidence, rust severity, and rust sporulation. The expression of genes coding for pathogenesis-related proteins increased positively with rust incidence in the inbred, while genes involved in homoeostasis and broader cell wall structuring processes were upregulated in the F1 hybrid. The enriched gene functions and associations revealed that a possible hypersensitive response (HR) in the inbred and a systemic acquired resistance (SAR) in the F1 hybrid were involved in the tolerance mechanisms to CLR stress. This is the first study to demonstrate the specific interactions between CLR and host at a molecular level, useful for identifying control targets for breeding perennial species.
Background. Coffee leaf rust (CLR) caused by Hemileia vastatrix Berk. & Br, is one of the most threatening diseases for Coffea arabica L. It is hypothesized that host tolerance to CLR relies on non-race-specific resistance genes.Methods. This study evaluated gene expression in leaves of two susceptible coffee cultivars (one inbred and one F1 hybrid) under different stress conditions: rust control (fungicide and untreated) and fruit thinning (thinned and un-thinned) treatments. RNA-seq analysis focused on the association of differentially expressed genes (DEGs) with CLR and associated the effect of the most significant genes into the phenotype, using regression and prediction statistical models.Results. Gene expression and gene ontology (GO) analysis allowed identification of 100 genes associated with quantitative traits. From these, 88 were correlated with rust incidence, rust severity, and rust sporulation. The expression of genes coding for pathogenesis-related proteins increased positively with rust incidence in the inbred, while genes involved in homoeostasis and broader cell wall structuring processes were upregulated in the F1 hybrid. The enriched gene functions and associations revealed that a possible hypersensitive response (HR) in the inbred and a systemic acquired resistance (SAR) in the F1 hybrid were involved in the tolerance mechanisms to CLR stress. This is the first study to demonstrate the specific interactions between CLR and host at a molecular level, useful for identifying control targets for breeding perennial species.