scholarly journals Maternal Overnutrition Programs Central Inflammation and Addiction-Like Behavior in Offspring

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Larisa Montalvo-Martínez ◽  
Roger Maldonado-Ruiz ◽  
Marcela Cárdenas-Tueme ◽  
Diana Reséndez-Pérez ◽  
Alberto Camacho
Keyword(s):  
Gene Expression ◽  
Fetal Programming ◽  
Gene Expression Pattern ◽  
Cell Types ◽  
Addictive Behavior ◽  
Adaptive Responses ◽  
Inflammatory Phenotype ◽  
Pregnancy And Lactation ◽  
Maternal Overnutrition ◽  
The Impact

Obesity or maternal overnutrition during pregnancy and lactation might have long-term consequences in offspring health. Fetal programming is characterized by adaptive responses to specific environmental conditions during early life stages. Programming alters gene expression through epigenetic modifications leading to a transgenerational effect of behavioral phenotypes in the offspring. Maternal intake of hypercaloric diets during fetal development programs aberrant behaviors resembling addiction in offspring. Programming by hypercaloric surplus sets a gene expression pattern modulating axonal pruning, synaptic signaling, and synaptic plasticity in selective regions of the reward system. Likewise, fetal programming can promote an inflammatory phenotype in peripheral and central sites through different cell types such as microglia and T and B cells, which contribute to disrupted energy sensing and behavioral pathways. The molecular mechanism that regulates the central and peripheral immune cross-talk during fetal programming and its relevance on offspring’s addictive behavior susceptibility is still unclear. Here, we review the most relevant scientific reports about the impact of hypercaloric nutritional fetal programming on central and peripheral inflammation and its effects on addictive behavior of the offspring.

scholarly journals Optimizing expression quantitative trait locus mapping workflows for single-cell studies

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anna S. E. Cuomo ◽  
Giordano Alvari ◽  
Christina B. Azodi ◽  
Davis J. McCarthy ◽  
Marc Jan Bonder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait Locus ◽  
Single Cell ◽  
Quantitative Trait ◽  
Cell Types ◽  
Expression Quantitative Trait Locus ◽  
Eqtl Mapping ◽  
Trait Locus ◽  
The Impact

Abstract Background Single-cell RNA sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states and promises to improve our understanding of genetic regulation across tissues in both health and disease. Results While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimize sc-eQTL mapping. Here, we evaluate the role of different normalization and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches. Conclusion We provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

scholarly journals Transcriptome analysis of the effect of AHR on productive and unproductive pathways of in vitro megakaryocytopoiesis

2021 ◽  
Author(s):  
Léa Malo ◽  
Valentin Do Sacramento ◽  
Christian Gachet ◽  
François Lanza ◽  
Henri de la Salle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptome Analysis ◽  
Gene Expression Pattern ◽  
Cell Types ◽  
Dependent Manner ◽  
Noticeable Effect ◽  
Human Cd34 ◽  
Extracellular Matrix Components ◽  
Genetic Perturbations

Human CD34+ progenitors can be differentiated in vitro into proplatelet-producing megakaryocytes (MKs) within 17 days. During this time, four cell populations emerge, phenotypically defined as CD34+CD41+ on day 7 (D7) and CD34+CD41+CD9- on D10 and D14 - qualified as productive because they can differentiate into proplatelet-forming cells during the D14-D17 period - and CD34-CD41+ or CD34+CD41+CD9+ on day 10 - qualified as unproductive because they are unable to form proplatelets later. Coculture with mesenchymal stem cells, or the presence of the AHR antagonist SR1, boosts the productive pathway in two ways: firstly, it increases the yield of D10 and D14 CD34+CD41+CD9- cells and secondly, it greatly increases their ability to generate proplatelets; in contrast, SR1 has no noticeable effect on the unproductive cell types. A transcriptome analysis was performed to decipher the genetic basis of these properties. This work represents the first extensive description of the genetic perturbations which accompany the differentiation of CD34+ progenitors into mature MKs at a subpopulation level. It highlights a wide variety of biological changes modulated in a time-dependent manner and allows anyone, according to his/her interests, to focus on specific biological processes accompanying MK differentiation. For example, the modulation of the expression of genes associated with cell proliferation, lipid and cholesterol synthesis, extracellular matrix components, intercellular interacting receptors and MK and platelet functions reflected the chronological development of the productive cells and pointed to unsuspected pathways. Surprisingly, SR1 only affected the gene expression profile of D10 CD34+CD41+CD9- cells; thus, as compared to these cells and those present on D14, the poorly productive D10 CD34+CD41+CD9- cells obtained in the absence of SR1 and the two unproductive populations present on D10 displayed an intermediate gene expression pattern. In other words, the ability to generate proplatelets between D10 and D14 appeared to be linked to the capacity of SR1 to delay MK differentiation, meanwhile avoiding intermediate and inappropriate genetic perturbations. Paradoxically, the D14 CD34+CD41+CD9- cells obtained under SR1- or SR1+ conditions were virtually identical, raising the question as to whether their strong differences in terms of proplatelet production, in the absence of SR1 and between D14 and D17, are mediated by miRNAs or by memory post-translational regulatory mechanisms.

scholarly journals Optimising expression quantitative trait locus mapping workflows for single-cell studies

2021 ◽  
Author(s):  
Anna S.E. Cuomo ◽  
Giordano Alvari ◽  
Christina B. Azodi ◽  
Davis J. McCarthy ◽  
Marc Jan Bonder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait Locus ◽  
Single Cell ◽  
Quantitative Trait ◽  
Cell Types ◽  
Expression Quantitative Trait Locus ◽  
Eqtl Mapping ◽  
Trait Locus ◽  
The Impact

AbstractSingle-cell RNA-sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states, and promises to improve our understanding of genetic regulation across tissues in both health and disease. While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimise sc-eQTL mapping. Here, we evaluate the role of different normalisation and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches and provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

131. SIRT3 IN OVARIAN CELLS IS ALTERED BY MATERNAL AGE AND OVARIAN RESERVE

2010 ◽  
Vol 22 (9) ◽  
pp. 49
Author(s):  
L. Pacella ◽  
D. Zander-Fox ◽  
T. Hussein ◽  
T. Fullston ◽  
M. Lane
Keyword(s):  
Gene Expression ◽  
Ovarian Reserve ◽  
Maternal Age ◽  
Cumulus Cells ◽  
Cell Types ◽  
Advanced Maternal Age ◽  
Cellular Functions ◽  
Protein Levels ◽  
Ovarian Cells ◽  
The Impact

Maternal age and reduced AMH levels affect the follicular environment and consequently oocyte viability. The Sirtuin family of protein deacetylases are able to regulate various cellular functions involved in the ageing process in other tissues. In particular, SIRT3 is related to longevity in several cell types and regulates mitochondrial function, however, its presence and role in ovarian cells remains unknown. This study therefore, investigated the presence of SIRT3 in granulosa and cumulus cells, from patients undergoing IVF, and determined the impact of maternal age and low AMH on SIRT3 levels. Granulosa and cumulus cells were collected from women (n = 36), after informed consent, and classified into 3 groups; A (<35 years, normal AMH), B (>40 years (advanced maternal age), normal AMH) and C (<35 years, low AMH). The presence of SIRT3 was determined by q-PCR (expressed as fold-change) or immunohistochemistry. SIRT3 was present in the ovarian cells of all patients analysed. SIRT3 gene expression was reduced in granulosa cells from women with low AMH (0.67 ± 0.17) compared to women with normal AMH (1.00 ± 0.23; P < 0.05). In cumulus cells, levels were reduced with advanced maternal age (0.81 ± 0.08) compared to women <35 years (1.00 ± 0.22; P < 0.05). SIRT3 protein co-localised with mitochondria in the ovarian cells, confirming previous findings for other cell types. In comparison to women <35 years with normal AMH, image analysis determined that SIRT3 protein levels were significantly reduced in the granulosa and cumulus cells from women of advanced maternal age by 21.4% and 31.8% and in women with low AMH by 34.1% and 47.2% respectively. This is the first study to demonstrate SIRT3 presence in human ovarian cells. The observation that SIRT3 levels are altered by advanced maternal age or low AMH (reduced ovarian reserve) implicate its role in ovarian ageing and plausibly in the decrease in oocyte viability observed in these women.

Impact of Trisomy 8 on Expression of Genes Located on Chromosome 8 in Different AML Subgroups.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2034-2034
Author(s):  
Claudia Schoch ◽  
Wolfgang Kern ◽  
Alexander Kohlmann ◽  
Martin Dugas ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Dosage ◽  
Gene Expression Pattern ◽  
Chromosome 8 ◽  
Support Vector ◽  
Trisomy 8 ◽  
Gene Dosage Effect ◽  
Class Prediction ◽  
Expression Of Genes ◽  
The Impact

Abstract Trisomy 8 is the most frequently observed trisomy in acute myeloid leukemia (AML). It occurs as a sole karyotype abnormality or in addition to other chromosome aberrations. It was the aim of this study to analyze the impact of trisomy 8 on the expression of genes located on chromosome 8 in different AML subgroups. Therefore, gene expression analyses were performed in a total of 567 AML cases using Affymetrix U133A+B oligonucleotide microarrays. The following 14 subgroups were analyzed: +8 sole (n=19), +8 within a complex aberrant karyotype (n=11), +8 with t(15;17) (n=7), +8 and inv(16) (n=3), +8 with t(8;21) (n=3), +8 and 11q23/MLL (n=8), and +8 with other abnormalities (n=10). These were compared to 200 AML with normal karyotype and the following subgroups without trisomy 8: complex aberrant karyotype (n=73), t(15;17) (n=36), inv(16) (n=46), t(8;21) (n=37), 11q23/MLL (n=37), and other abnormalities (n=77). In total 1188 probe sets cover sequences located on chromosome 8 representing 580 genes. A significant higher mean expression of all genes located on chromosome 8 was observed in subgroups with +8 in comparison to their respective control groups (for all comparisons, p<0.05). Significantly higher expressed genes in groups with +8 in comparison to the respective groups without +8 were identified in all comparisons. The number of identified genes ranged from 40 in 11q23/MLL to 326 in trisomy 8 sole vs. normal. There was no common gene significantly overexpressed in all comparisons. Three genes (TRAM1, CHPPR, MGC40214) showed a significantly higher expression in 5 out of 7 comparisons. Between 19 and 107 genes with an exclusive overexpression in trisomy 8 cases in only one subtype comparison were identified. In addition, we performed class prediction using support vector machines (SVM) including all probe sets on the arrays. In one approach all 14 different subgroups were analyzed as one class each. Only 3 out of 61 cases with trisomy 8 were assigned into their correct subclass, while 40 cases were assigned to their corresponding genetic subclass without trisomy 8. In a second approach only two classes were defined: all cases with trisomy 8 combined vs. all cases without trisomy 8. Only 26 out of 61 (42.6%) with trisomy 8 were identified correctly underlining the fact that no distinct gene expression pattern is associated with trisomy 8 in general. Performing SVM only with genes located on chromosome 8 did not improve the correct assignment of cases with trisomy 8 overall. Only cases with trisomy 8 sole were correctly predicted in 58% as compared to 11% in SVM using all genes. In conclusion, overall the gain of chromosome 8 leads to a higher expression of genes located on chromosome 8. However, no consistent pattern of genes was identified which shows a higher expression in all AML subtypes with trisomy 8. This data suggest that the higher expression of genes located on chromosome 8 only in part is directly related to a gene dosage effect. Trisomy 8 may rather provide a platform for a higher expression of chromosome 8 genes which are specifically upregulated by accompanying genetic abnormalities in the respective AML subtypes. Therefore, trisomy 8 does not seem to be an abnormality determining specific disease characteristics such as the well known primary aberrations (t(8;21), inv(16), t(15;17), MLL/11q23) but rather a disease modulating secondary event in addition to primary cytogenetic or moleculargenetic aberrations.

scholarly journals Compared to Australian Cultivars, European Summer Wheat (Triticum aestivum) Overreacts When Moderate Heat Stress Is Applied at the Pollen Development Stage

Agronomy ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 99 ◽  
Author(s):  
Kevin Begcy ◽  
Anna Weigert ◽  
Andrew Egesa ◽  
Thomas Dresselhaus
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Heat Waves ◽  
Negative Impact ◽  
Pollen Viability ◽  
Wheat Yield ◽  
Gene Expression Pattern ◽  
Type A ◽  
Transcriptional Level ◽  
The Impact

Heat stress frequently imposes a strong negative impact on vegetative and reproductive development of plants leading to severe yield losses. Wheat, a major temperate crop, is more prone to suffer from increased temperatures than most other major crops. With heat waves becoming more intense and frequent, as a consequence of global warming, a decrease in wheat yield is highly expected. Here, we examined the impact of a short-term (48 h) heat stress on wheat imposed during reproduction at the pollen mitosis stage both, at the physiological and molecular level. We analyzed two sets of summer wheat germplasms from Australia (Kukri, Drysdale, Gladius, and RAC875) and Europe (Epos, Cornetto, Granny, and Chamsin). Heat stress strongly affected gas exchange parameters leading to reduced photosynthetic and transpiration rates in the European cultivars. These effects were less pronounced in Australian cultivars. Pollen viability was also reduced in all European cultivars. At the transcriptional level, the largest group of heat shock factor genes (type A HSFs), which trigger molecular responses as a result of environmental stimuli, showed small variations in gene expression levels in Australian wheat cultivars. In contrast, HSFs in European cultivars, including Epos and Granny, were strongly downregulated and partly even silenced, while the high-yielding variety Chamsin displayed a strong upregulation of type A HSFs. In conclusion, Australian cultivars are well adapted to moderate heat stress compared to European summer wheat. The latter strongly react after heat stress application by downregulating photosynthesis and transpiration rates as well as differentially regulating HSFs gene expression pattern.

scholarly journals Identification and expression analysis of zebrafish testis-specific gene 10 (tsga10)

2019 ◽  
Vol 63 (11-12) ◽  
pp. 623-629
Author(s):  
Shohreh Asghari-Givehchi ◽  
Mohammad Hossein-Modarressi
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Developmental Stages ◽  
Hypoxia Inducible Factor ◽  
Gene Expression Pattern ◽  
Model Organism ◽  
Orthologous Gene ◽  
Cell Types ◽  
Specific Gene ◽  
Cell Stage

Several clinical studies suggest that testis-specific gene antigen 10 (TSGA10) is a cancer-testis antigen with a discernible expression pattern in the testis. Recent studies have highlighted that TSGA10 overexpression in HeLa cells impairs the transcriptional activity of hypoxia-inducible factor alpha (HIF-1&alpha;) and inhibits angiogenesis. In this study, we used the zebrafish as a powerful model organism to identify and characterize the orthologue of TSGA10. We analyzed the gene expression pattern by RT-PCR and whole mount in situ hybridization and overexpressed the tsga10 protein by mRNA microinjection. Our results revealed that during early development, tsga10 expression is enriched, but gradually subsides between 0 and 72 hours post fertilization (hpf). There was no detectable transcript at the larval stages. In adult fish, we found high expression levels of tsga10 in the testis and unfertilized egg and low levels of gene expression in the brain, eyes and muscle. Overexpression of tsga10, using tsga10 mRNA microinjection into one-cell stage embryos, resulted in angiogenic and morphological defects at 24 and 48 hpf. This study clarified the expression pattern of tsga10 in different developmental stages and adult tissues, suggesting that tsga10 may have a related biological role in different cell types and tissues. Our results indicate that tsga10 mRNA at embryonic stages is maternally deposited, indicating a transient functional role during embryogenesis. Our findings suggest that tsga10 is a human orthologous gene relevant for future studies to elucidate its mechanism of action in angiogenesis.

scholarly journals Killing from the inside: Intracellular role of T3SS in the fate ofPseudomonas aeruginosawithin macrophages revealed bymgtCandoprFmutants

2018 ◽  
Author(s):  
Preeti Garai ◽  
Laurence Berry ◽  
Malika Moussouni ◽  
Sophie Bleves ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Gene Expression Pattern ◽  
Type Iii Secretion System ◽  
Cell Types ◽  
Cell Lysis ◽  
Secretion System ◽  
Intracellular Bacteria ◽  
Type Iii

AbstractWhile considered solely an extracellular pathogen, increasing evidence indicates thatPseudomonas aeruginosaencounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-typeP. aeruginosaPAO1 strain by live and electron microscopy.P. aeruginosafirst resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis. Two bacterial factors, MgtC and OprF, recently identified to be important for survival ofP. aeruginosain macrophages, were found to be involved in bacterial escape from the phagosome as well as cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes ofP. aeruginosawere down-regulated within macrophages in bothmgtCandoprFmutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern ofmgtCandoprFmutants, a T3SS mutant(ΔpscN)exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellularP. aeruginosatowards macrophages. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life ofP. aeruginosa, allowing internalized bacteria to evade macrophages.Author summaryThe ability of professional phagocytes to ingest and kill microorganisms is central to host defense andPseudomonas aeruginosahas developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen,P. aeruginosahas been reported to be engulfed by macrophages in animal models. Here, we visualized the fate ofP. aeruginosawithin cultured macrophages, revealing macrophage lysis driven by intracellularP. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival ofP. aeruginosa, appeared to play role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly inmgtCandoprFmutants. We further showed that intramacrophageP. aeruginosauses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage ofP. aeruginosathat could contribute to bacterial dissemination.

scholarly journals c-Kit modifies the inflammatory status of smooth muscle cells

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3418 ◽  
Author(s):  
Lei Song ◽  
Laisel Martinez ◽  
Zachary M. Zigmond ◽  
Diana R. Hernandez ◽  
Roberta M. Lassance-Soares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Dependent Manner ◽  
Functional Assays ◽  
Inflammatory Status ◽  
Inflammatory Phenotype ◽  
Pathway Regulation

Backgroundc-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis.MethodsHigh-throughput microarray assays andin silicopathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations.ResultsThe microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner.DiscussionOur study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

scholarly journals Partial reprogramming restores youthful gene expression through transient suppression of cell identity

2021 ◽  
Author(s):  
Antoine Roux ◽  
Chunlian Zhang ◽  
Jonathan Paw ◽  
José-Zavalara Solorio ◽  
Twaritha Vijay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Mammalian Cells ◽  
Cell Types ◽  
Alternative Strategies ◽  
Cell Identity ◽  
Pluripotency Factors ◽  
Reprogramming Factors ◽  
The Impact ◽  
Yamanaka Factor

Transient induction of pluripotent reprogramming factors has been reported to reverse some features of aging in mammalian cells and tissues. However, the impact of transient reprogramming on somatic cell identity programs and the necessity of individual pluripotency factors remain unknown. Here, we mapped trajectories of transient reprogramming in young and aged cells from multiple murine cell types using single cell transcriptomics to address these questions. We found that transient reprogramming restored youthful gene expression in adipocytes and mesenchymal stem cells but also temporarily suppressed somatic cell identity programs. We further screened Yamanaka Factor subsets and found that many combinations had an impact on aging gene expression and suppressed somatic identity, but that these effects were not tightly entangled. We also found that a transient reprogramming approach inspired by amphibian regeneration restored youthful gene expression in aged myogenic cells. Our results suggest that transient pluripotent reprogramming poses a neoplastic risk, but that restoration of youthful gene expression can be achieved with alternative strategies.

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