RESUMEN
Combinational antiretroviral therapy (cART) suppresses human immunodeficiency virus type 1 (HIV-1) viral replication and pathogenesis in acquired immunodeficiency syndrome (AIDS) patients. However, HIV-1 remains in the latent stage of infection by suppressing viral transcription, which hinders an HIV-1 cure. One approach for an HIV-1 cure is the "shock and kill" strategy. The strategy focuses on reactivating latent HIV-1, inducing the viral cytopathic effect and facilitating the immune clearance for the elimination of latent HIV-1 reservoirs. Here, we reported that the H3K4 trimethylation (H3K4me3)-specific demethylase KDM5A/B play a role in suppressing HIV-1 Tat/LTR-mediated viral transcription in HIV-1 latent cells. Furthermore, we evaluated the potential of KDM5-specific inhibitor JQKD82 as an HIV-1 "shock and kill" agent. Our results showed that JQKD82 increases the H3K4me3 level at HIV-1 5' LTR promoter regions, HIV-1 reactivation, and the cytopathic effects in an HIV-1-latent T cell model. In addition, we identified that the combination of JQKD82 and AZD5582, a non-canonical NF-κB activator, generates a synergistic impact on inducing HIV-1 lytic reactivation and cell death in the T cell. The latency-reversing potency of the JQKD82 and AZD5582 pair was also confirmed in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 aviremic patients and in an HIV-1 latent monocyte. In latently infected microglia (HC69) of the brain, either deletion or inhibition of KDM5A/B results in a reversal of the HIV-1 latency. Overall, we concluded that KDM5A/B function as a host repressor of the HIV-1 lytic reactivation and thus promote the latency and the survival of HIV-1 infected reservoirs.
Asunto(s)
Infecciones por VIH , VIH-1 , Activación Viral , Latencia del Virus , VIH-1/fisiología , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Latencia del Virus/efectos de los fármacos , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , Activación Viral/efectos de los fármacos , Proteína 2 de Unión a Retinoblastoma/metabolismo , Proteína 2 de Unión a Retinoblastoma/genética , Infección Latente/virología , Replicación Viral/efectos de los fármacos , Duplicado del Terminal Largo de VIH/genética , Supervivencia Celular , Línea Celular , Histonas/metabolismo , Proteínas Nucleares , Proteínas Represoras , Histona Demetilasas con Dominio de JumonjiRESUMEN
Background: Malnutrition is identified as a risk-factor for insufficient polioseroconversion in the context of a vaccine-derived polio virus (VDPV) outbreak prone region. To assess the prevalence of malnutrition and its link to poliovirus insufficient immunity, a cross-sectional household survey was conducted in the regions of Haut- Lomami and Tanganyika, DRC. Methods: In March 2018, we included 968 healthy children aged 6 to 59 months from eight out of 27 districts. Selection of study locations within these districts was done using a stratified random sampling method, where villages were chosen based on habitat characteristics identified from satellite images. Consent was obtained verbally in the preferred language of the participant (French or Swahili) by interviewers who received specific training for this task. Furthermore, participants contributed a dried blood spot sample, collected via finger prick. To assess malnutrition, we measured height and weight, applying WHO criteria to determine rates of underweight, wasting, and stunting. The assessment of immunity to poliovirus types 1, 2, and 3 through the detection of neutralizing antibodies was carried out at the CDC in Atlanta, USA. Results: Of the study population, we found 24.7% underweight, 54.8% stunted, and 15.4% wasted. With IC95%, underweight (OR=1.50; [1.11-2.03]), and the non-administration of vitamin A (OR=1.96; [1.52-2.54]) were significantly associated with seronegativity to polioserotype 1. Underweight (OR=1.64; [1.20-2.24]) and the non-administration of vitamin A (OR=1.55; [1.20-2.01]) were significantly associated with seronegativity to polioserotype 2. Underweight (OR=1.50; [1.11-2.03]), and the non-administration of vitamin A (OR=1.80. [1.38-2.35]) were significantly associated with seronegativity to polioserotype 3. Underweight (OR=1.68; IC95% [1.10-2.57]) and the non-administration of vitamin A (OR=1.82; IC95% [1.30-2.55]) were significantly associated with seronegativity to all polioserotypes. Conclusion: This study reveals a significant association between underweight and polioseronegativity in children. In order to reduce vaccine failures in high-risk areas, an integrated approach by vaccination and nutrition programs should be adopted.
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A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.
Asunto(s)
Lignina , Estilbenos , Lignina/metabolismo , Éter , Agrobacterium/metabolismo , Éteres/química , Éteres de Etila , Glutatión/metabolismoRESUMEN
Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.
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Carotenoides , Solanum lycopersicum , Carotenoides/metabolismo , Solanum lycopersicum/genética , Reprogramación Metabólica , Plantas/metabolismo , HomeostasisRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.
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COVID-19 , Exorribonucleasas , IMP Deshidrogenasa , FN-kappa B , Proteínas no Estructurales Virales , Bortezomib , Citocinas/metabolismo , Exorribonucleasas/metabolismo , Humanos , IMP Deshidrogenasa/metabolismo , Inosina , Interleucina-6 , Interleucina-8 , Ácido Micofenólico , FN-kappa B/metabolismo , Oxidorreductasas , Proteómica , Ribavirina , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismoRESUMEN
FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.
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Proteínas de Ciclo Celular/metabolismo , Células Epiteliales/metabolismo , Interferones/metabolismo , Células Asesinas Naturales/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , COVID-19 , Proteínas de Unión al ADN/genética , Células Epiteliales/inmunología , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Células Asesinas Naturales/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , SARS-CoV-2 , Factores de Elongación Transcripcional/genética , Virus Zika/metabolismo , Infección por el Virus ZikaRESUMEN
Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20-fold and the triacylglycerol content increases by around 30-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.
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Arabidopsis , Sistemas CRISPR-Cas , Arabidopsis/genética , Arabidopsis/metabolismo , Edición Génica , Fusión Génica , Genómica , TransgenesRESUMEN
Polyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposi's sarcoma-associated Herpesvirus (KSHV), a defined human oncogenic virus, leads to profound alterations of host metabolic landscape to favor development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection through modulation of key enzymes (ODC1 and DHPS) of these pathways. During KSHV latency, ODC1 and DHPS are upregulated along with increase of hypusinated eIF5A (hyp-eIF5A), while hyp-eIF5A is further induced along with reduction of ODC1 and intracellular polyamines during KSHV lytic reactivation. In return these metabolic pathways are required for both KSHV lytic reactivation and de novo infection. Further analysis unraveled that synthesis of critical KSHV latent and lytic proteins (LANA, RTA) depends on hypusinated-eIF5A. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by inhibitors targeting these pathways. Collectively, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination pathways promote viral propagation, thus defining new therapeutic targets to treat KSHV-associated malignancies.
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Herpesvirus Humano 8 , Sarcoma de Kaposi , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Humanos , Poliaminas/metabolismo , Activación Viral/genética , Latencia del Virus/genética , Replicación ViralRESUMEN
Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a â¼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.
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Ácidos Cumáricos , Semillas , Colina/análogos & derivados , Colina/metabolismo , Ácidos Cumáricos/metabolismo , Semillas/metabolismoRESUMEN
Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.
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Proteínas de Ciclo Celular/metabolismo , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Línea Celular , Infecciones por Virus de Epstein-Barr , Infecciones por VIH , Humanos , Quinasa Tipo Polo 1RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.
RESUMEN
FACT ( FA cilitates C hromatin T ranscription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.
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EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses.
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Biomarcadores/metabolismo , Epigénesis Genética , Gammaherpesvirinae/aislamiento & purificación , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/complicaciones , Interacciones Huésped-Patógeno , Neoplasias Gástricas/genética , Biomarcadores/análisis , Metilación de ADN , Gammaherpesvirinae/genética , Células HEK293 , Infecciones por Herpesviridae/virología , Proteínas Homeobox A10/genética , Humanos , Incidencia , Metalotioneína/genética , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virología , Activación Viral , Replicación ViralRESUMEN
Virus contamination of water is a threat to human health in many countries. Current solutions for inactivation of viruses mainly rely on environmentally burdensome chemical oxidation or energy-intensive ultraviolet irradiation, which may create toxic secondary products. Here, we show that renewable plant biomass-sourced colloidal lignin particles (CLPs) can be used as agglomeration agents to facilitate removal of viruses from water. We used dynamic light scattering (DLS), electrophoretic mobility shift assay (EMSA), atomic force microscopy and transmission electron microscopy (AFM, TEM), and UV spectrophotometry to quantify and visualize adherence of cowpea chlorotic mottle viruses (CCMVs) on CLPs. Our results show that CCMVs form agglomerated complexes with CLPs that, unlike pristine virus particles, can be easily removed from water either by filtration or centrifugation. Additionally, cationic particles formed by adsorption of quaternary amine-modified softwood kraft lignin on the CLPs were also evaluated to improve the binding interactions with these anionic viruses. We foresee that due to their moderate production cost, and high availability of lignin as a side-stream from biorefineries, CLPs could be an alternative water pretreatment material in a large variety of systems such as filters, packed columns, or flocculants.
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Seeds exhibit wide variation in the fatty acid composition of their storage oil. However, the genetic basis of this variation is only partially understood. Here we have used a multi-parent advanced generation inter-cross (MAGIC) population to study the genetic control of fatty acid chain length in Arabidopsis thaliana seed oil. We mapped four quantitative trait loci (QTL) for the quantity of the major very long chain fatty acid species 11-eicosenoic acid (20:1), using multiple QTL modelling. Surprisingly, the main-effect QTL does not coincide with FATTY ACID ELONGASE 1 and a parallel genome wide association study suggested that LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE 2 (LPCAT2) is a candidate for this QTL. Regression analysis also suggested that LPCAT2 expression and 20:1 content in seeds of the 19 MAGIC founder accessions are related. LPCAT is a key component of the Lands cycle; an acyl editing pathway that enables acyl-exchange between the acyl-Coenzyme A and phosphatidylcholine precursor pools used for microsomal fatty acid elongation and desaturation, respectively. We Mendelianised the main-effect QTL using biparental chromosome segment substitution lines and carried out complementation tests to show that a single cis-acting polymorphism in the LPCAT2 promoter causes the variation in seed 20:1 content, by altering the LPCAT2 expression level and total LPCAT activity in developing siliques. Our work establishes that oilseed species exhibit natural variation in the enzymic capacity for acyl editing and this contributes to the genetic control of storage oil composition.
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Arabidopsis/genética , Ácidos Grasos/metabolismo , Aceites de Plantas/metabolismo , Semillas/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos/química , Ácidos Grasos/genética , Ácidos Grasos Monoinsaturados/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Estudio de Asociación del Genoma Completo , Aceites de Plantas/química , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo , Semillas/metabolismoRESUMEN
In clinical organ transplantation, flow cytometry crossmatching can be performed on total blood with a hemolysis step or after a preliminary mononuclear cell separation step using a standard Ficoll-Hypaque protocol. Here, we compared the Ficoll-Hypaque step with a faster technique for isolating mononuclear cells (the SepMate tube), using the same samples (collected and stored at room temperature for 0, 24, 48 or 72 hours). We found that the SepMate separation protocol is easily applied to flow cytometry crossmatching (with or without pronase treatment), provided that the samples have been stored at room temperature for 48 hours or less. Conversely, the Ficoll-Hypaque protocol should be used if the samples have been stored for more than 48 hours at room temperature.
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Separación Celular/métodos , Citometría de Flujo/métodos , Prueba de Histocompatibilidad/métodos , Leucocitos Mononucleares/citología , Linfocitos B/citología , Fluorescencia , Humanos , Linfocitos T/citologíaRESUMEN
WHAT IS KNOWN AND OBJECTIVE: Voriconazole and vincristine are major therapeutics in paediatric haematology. However, the risk-benefit ratio of the treatment of invasive aspergillosis with voriconazole in patients receiving vincristine-based chemotherapy remains unclear. CASE DESCRIPTION: We report severe peripheral and central neurological disorders in a 14-year-old girl with T-cell acute lymphoblastic leukaemia and pulmonary aspergillosis. The case describes a strong exacerbation by voriconazole of the vincristine-induced neuropathic pains. It shows the high variability of the trough serum concentration of voriconazole leading to antifungal treatment failure and suggests that its own central neurotoxicity could also be potentiated by vincristine. WHAT IS NEW AND CONCLUSION: Given the risk of either insufficient antifungal efficacy or excessive neurological disorders, this case warns on a probable unfavourable risk-benefit profile of voriconazole during vincristine-based chemotherapy in adolescents.
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Aspergilosis/tratamiento farmacológico , Enfermedades del Sistema Nervioso/inducido químicamente , Vincristina/efectos adversos , Vincristina/uso terapéutico , Voriconazol/efectos adversos , Voriconazol/uso terapéutico , Adolescente , Antifúngicos/efectos adversos , Antifúngicos/uso terapéutico , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/uso terapéutico , Femenino , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.
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Acuicultura , Carotenoides/biosíntesis , Ingeniería Metabólica/métodos , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Pigmentación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.