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1.
Signal Transduct Target Ther ; 8(1): 44, 2023 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-36710358

RESUMEN

Radiochemotherapy (RCT) is a powerful treatment for cervical cancer, which affects not only malignant cells but also the immune and stromal compartments of the tumor. Understanding the remodeling of the local ecosystem induced by RCT would provide valuable insights into improving treatment strategies for cervical cancer. In this study, we applied single-cell RNA-sequencing to paired pre- and post-RCT tumor biopsies from patients with cervical cancer and adjacent normal cervical tissues. We found that the residual population of epithelial cells post-RCT showed upregulated expression of MHC class II genes. Moreover, RCT led to the accumulation of monocytic myeloid-derived suppressor cells with increased pro-inflammatory features and CD16+ NK cells with a higher cytotoxic gene expression signature. However, subclusters of T cells showed no significant increase in the expression of cytotoxic features post-RCT. These results reveal the complex responses of the tumor ecosystem to RCT, providing evidence of activation of innate immunity and MHC-II upregulation in cervical cancer.


Asunto(s)
Antineoplásicos , Neoplasias del Cuello Uterino , Femenino , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/radioterapia , Regulación hacia Arriba/genética , Inmunidad Innata/genética , Quimioradioterapia/métodos , ARN
2.
Development ; 150(2)2023 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-36715566

RESUMEN

A hallmark of all germ cells is the presence of germ granules: assemblies of proteins and RNA that lack a delineating membrane and are proposed to form via condensation. Germ granules across organisms share several conserved components, including factors required for germ cell fate determination and maintenance, and are thought to be linked to germ cell development. The molecular functions of germ granules, however, remain incompletely understood. In this Development at a Glance article, we survey germ granules across organisms and developmental stages, and highlight emerging themes regarding granule regulation, dynamics and proposed functions.


Asunto(s)
Caenorhabditis elegans , Gránulos de Ribonucleoproteína de Células Germinales , Animales , Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Gránulos Citoplasmáticos/metabolismo
3.
Methods Mol Biol ; 2626: 323-333, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36715913

RESUMEN

The production of eggs in the Drosophila ovary requires complex interactions between multiple cell types that coexist within the same solid tissue. This cellular heterogeneity makes the ovary a rich subject of study, but also makes it challenging to identify transcriptional differences between individual cell types using methods such as bulk RNA sequencing. The development of single-cell RNA sequencing (scRNA-seq) techniques addresses this limitation by providing an avenue to profile genetic and functional heterogeneity at a cellular resolution. Here, we describe the isolation and preparation of the Drosophila ovary for scRNA-seq. This protocol emphasizes a short preparation time, high cell viability, prevention of RNA-degradation, and reduction of technical variation to achieve highly reproducible single-cell profiles.


Asunto(s)
Drosophila , Análisis de la Célula Individual , Animales , Femenino , Drosophila/genética , Análisis de la Célula Individual/métodos , Análisis de Secuencia de ARN/métodos , Ovario/metabolismo , Secuencia de Bases , ARN/genética , ARN/metabolismo , Perfilación de la Expresión Génica/métodos
4.
Nature ; 613(7944): 582-587, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36599980

RESUMEN

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.


Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , ARN , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/metabolismo , ADN/química , ADN/inmunología , ADN/metabolismo , ARN/química , ARN/metabolismo , Activación Enzimática , Dominio Catalítico , Especificidad por Sustrato
5.
Nature ; 613(7944): 588-594, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36599979

RESUMEN

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR-Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3-5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.


Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , ADN , ARN , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , ADN de Cadena Simple/metabolismo , ARN/metabolismo , Respuesta SOS en Genética , Daño del ADN , Edición Génica
6.
Cell Res ; 33(1): 9-10, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36588116

Asunto(s)
ARN
7.
Int J Oral Sci ; 15(1): 2, 2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36596771

RESUMEN

Saliva testing is a vital method for clinical applications, for its noninvasive features, richness in substances, and the huge amount. Due to its direct anatomical connection with oral, digestive, and endocrine systems, clinical usage of saliva testing for these diseases is promising. Furthermore, for other diseases that seeming to have no correlations with saliva, such as neurodegenerative diseases and psychological diseases, researchers also reckon saliva informative. Tremendous papers are being produced in this field. Updated summaries of recent literature give newcomers a shortcut to have a grasp of this topic. Here, we focused on recent research about saliva biomarkers that are derived from humans, not from other organisms. The review mostly addresses the proceedings from 2016 to 2022, to shed light on the promising usage of saliva testing in clinical diagnostics. We recap the recent advances following the category of different types of biomarkers, such as intracellular DNA, RNA, proteins and intercellular exosomes, cell-free DNA, to give a comprehensive impression of saliva biomarker testing.


Asunto(s)
Exosomas , Saliva , Humanos , Saliva/metabolismo , Biomarcadores/metabolismo , ARN , Exosomas/metabolismo
8.
AIDS Res Ther ; 20(1): 1, 2023 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-36597160

RESUMEN

OBJECTIVES: We assessed the virologic efficacy of switching to co-formulated elvitegravir, cobicistat, emtricitabine, tenofovir disoproxil fumarate (E/C/F/TDF) in patients with controlled HIV infection. METHODS: We conducted a retrospective multicenter observational cohort study including adult patients with controlled HIV-1 infection on any stable antiretroviral (ART) regimen, who switched to E/C/F/TDF. Success was measured by the proportion of patients with plasma viral load < 50 copies/ml at W48 using the FDA snapshot algorithm. We also assessed risk factors associated with virological failure (VF). RESULTS: 382 patients with HIV RNA < 50 copies/mL who switched to E/C/F/TDF were included in the study. Most patients (69.9%) were male, with median age 44 years (IQR 38-51), who had been on ART for a median of 7 years (IQR 4-13). Median CD4 count was 614/mm3 and 24.6% of the patients had a history of previous virological failure. The reasons for switching were simplification (67.0%) and tolerance issues (22.0%). At week 48, 314 (82.0% [95% CI 78.4-86.0]) patients had HIV RNA < 50 copies/mL, 13 (3.5% [95% CI 3.64-8.41]) experienced virological failure. Genotype at failure was available in 6/13 patients with detection of resistance-associated mutations to integrase inhibitors and NRTIs in 5/6 (83.3%) patients. We found no predictive factor associated with virological failure except for a borderline significance with the duration of viral suppression before the switch. Tolerability of E/C/F/TDF was good with 23/382 (6.0%) patients experiencing mild adverse reactions. CONCLUSION: In our cohort, switching well-suppressed patients to E/C/F/TDF resulted in few virologic failures and was well tolerated. However, resistance to integrase inhibitors emerged in patients with virological failure.


Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Adulto , Humanos , Masculino , Femenino , Infecciones por VIH/tratamiento farmacológico , Tenofovir/uso terapéutico , Tenofovir/efectos adversos , Emtricitabina/uso terapéutico , Emtricitabina/efectos adversos , Cobicistat/uso terapéutico , Cobicistat/efectos adversos , Fármacos Anti-VIH/efectos adversos , Inhibidores de Integrasa/uso terapéutico , Estudios de Cohortes , ARN
9.
Arch Virol ; 168(1): 29, 2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36598610

RESUMEN

The cotton boll weevil (CBW; Anthonomus grandis; Coleoptera: Curculionidae) is considered the major insect pest of cotton, causing considerable losses in yield and fiber quality. An increase in the boll weevil population due to increasingly inefficient chemical control measures is of great concernamong cotton producers. The absence of conventional or transgenic cultivars with minimal resistance to CBW has stimulated the search for new molecular and biological tools for efficient control of this insect pest. In this study, we used a metagenomic approach based on RNA deep sequencing to investigate the presence of viruses and coding viral RNA in apparently healthy native adult CBW insects collected from cotton crops in Mato Grosso state, Brazil. Using an Illumina HiSeq 2000 paired-end platform, 138,798 virus-related reads were obtained, and a consensus sequence of a putative new virus, 10,632 nucleotides in length, was assembled. The sequences of the 5' and 3' untranslated regions (UTRs) were determined by rapid amplification of cDNA ends (RACE), followed by Nanopore sequencing. The complete genome sequence included a 5'-UTR (1,158 nucleotides), a 3'-UTR (561 nucleotides), and a single ORF of 8,913 nucleotides encoding a large polyprotein. Sequence analysis of the putative polyprotein showed several regions with high sequence similarity to structural and non-structural proteins of viruses of the family Iflaviridae. Pairwise alignments of polyprotein amino acid sequences showed the highest sequence identity (32.13%) to a partial polyprotein sequence of a putative iflavirus (QKN89051.1) found in samples from wild zoo birds in China. Phylogenetic analysis based on full polyprotein sequences of different iflaviruses indicated that this new picorna-like virus is most closely related to iflaviruses found in lepidopteran insects, and it was therefore tentatively named "Anthonomus grandis iflavirus 1" (AgIV-1). This is, to our knowledge, the first complete viral genome sequence found in CBW, and it could provide a basis for further studies about the infectivity and transmission of this virus and its possible association with symptoms or acute disease. AgIV-1 could potentially be used to develop biological or molecular tools, such as a viral vector to carry interfering RNA molecules for CBW control.


Asunto(s)
Escarabajos , Virus , Gorgojos , Animales , Filogenia , Virus/genética , Nucleótidos , ARN , Gossypium
10.
PLoS Pathog ; 19(1): e1011049, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36603036

RESUMEN

The arenavirus nucleoprotein (NP) plays an important role in the virus' ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-ß promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-ß mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.


Asunto(s)
Arenavirus , Arenavirus/genética , Interferones , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Quinasa I-kappa B , Exonucleasas/genética , ARN
11.
AIDS Res Ther ; 20(1): 3, 2023 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-36604746

RESUMEN

BACKGROUND: Treatment management after repeated failure of antiretroviral therapy (ART) is difficult due to resistance and adherence challenges. For people who have failed non-nucleoside reverse transcriptase inhibitor-(NNRTI-) and protease inhibitor-(PI-) based regimens with no or limited resistance, remaining on PI-based ART is an option. Using data from an ART strategy trial (A5288) in low/middle-income countries which included this option, we explored whether predictors can be identified distinguishing those who experienced further virologic failure from those who achieved and maintained virologic suppression. METHODS: A5288 enrolled people with confirmed HIV-1 RNA ≥ 1000 copies/mL after ≥ 24 weeks of PI-based ART and prior failure on NNRTI-based ART. This analysis focused on the 278 participants with no resistance to the PI being taken and no or limited nucleoside reverse transcriptase inhibitor (NRTI) resistance, who continued their PI with flexibility to change NRTIs. Proportional hazards models were used to evaluate predictors of virologic failure during follow-up (VF: confirmed HIV-1 RNA ≥ 1000 copies/mL at ≥ 24 weeks of follow-up). RESULTS: 56% of participants were female. At study entry, median age was 40 years, time on ART 7.8 years, CD4 count 169 cells/mm3, HIV-1 RNA 20,444 copies/mL; and 37% had NRTI resistance. The estimated proportion experiencing VF increased from 39% at week 24 to 60% at week 96. In multivariable analysis, significant predictors at study entry of VF were higher HIV-1 RNA (adjusted hazard ratio: 2.20 for ≥ 10,000 versus < 10,000 copies/mL), lower age (1.96 for < 30 versus ≥ 30 years), NRTI resistance (1.74 for present versus absent), lower CD4 count (1.73 for < 200 versus ≥ 200 cells/mm3), and shorter ART duration (1.62 for < 10 versus ≥ 10 years). There was a strong trend in proportion with VF at week 96 with the number of these five risk factors that a participant had, varying from 8% for zero, to 31%, 40%, 73%, and 100% for one, two, three, and four/five. Only 13% of participants developed new NRTI or PI resistance mutations. CONCLUSION: A simple count of five predictors might have value for identifying risk of continued VF. Novel antiretroviral and adherence support interventions are needed to improve virologic outcomes for higher risk individuals.


Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Humanos , Femenino , Adulto , Masculino , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Inhibidores de la Transcriptasa Inversa/efectos adversos , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Terapia Antirretroviral Altamente Activa , Antirretrovirales/uso terapéutico , Recuento de Linfocito CD4 , Carga Viral , ARN , Resultado del Tratamiento
12.
Hum Exp Toxicol ; 42: 9603271221149656, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36607285

RESUMEN

BACKGROUND: Hyperglycemia is closely related to adverse pregnancy outcomes including pre-eclampsia (PE), a life-threatening complication with a substantial morbidity and mortality. However, the pathogenesis of abnormal placentation in gestational diabetes mellitus (GDM)-associated PE remains elusive. METHOD: Here we isolated exosomes from the human umbilical vein endothelial cells (HUVECs) treated with normal level of glucose (NG) and high levels of glucose (HG). The exosomes were added to HTR-8a/SVneo cells, a trophoblast cell line. High-throughput RNA-sequencing was performed to analyzed the changed RNAs in the exosomes and exosome-treated HTR-8a/SVneo cells. HTR-8a/SVneo cell phenotypes were evaluated from the aspects of cell proliferation, cell invasion and DNA damage. RESULTS: After treatment with HG, the changed RNAs in exosomes was enriched in RNA stabilization and oxidative stress. The altered RNAs in the HTR-8a/SVneo cells treated with exosomes from HG-induced HUVECs were enriched in pathways related to cell adhesion, migration, DNA damage response and angiogenesis. The HG-induced exosomes impaired the proliferation and invasion of HTR-8a cells and caused the DNA damage. HG up-regulated PUM2 in the exosomes and exosome-treated HTR-8a/SVneo cells. PUM2 interacted with SOX2 mRNA, resulting in the mRNA degradation. Overexpression of SOX2 prevented the damage to HTR-8a/SVneo cells caused by the exosomes from HG-induced HUVECs. CONCLUSIONS: We demonstrate that high glucose-induced endothelial exosomes mediate abnormal phenotypes of trophoblasts through PUM2-mediated repression of SOX2. Our results reveal a novel regulatory mechanism of hyperglycemia in development of abnormal placentation and provide potential targets for preventing adverse pregnancy outcomes.


Asunto(s)
Exosomas , Hiperglucemia , Preeclampsia , Embarazo , Femenino , Humanos , Placentación , Trofoblastos , Exosomas/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Hiperglucemia/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Preeclampsia/genética , Glucosa/farmacología , ARN/metabolismo , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo
13.
Curr Protoc ; 3(1): e640, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36607644

RESUMEN

This article describes high-yield procedures for protection of purine ribonucleosides based on a reaction that allows highly regioselective 2'-silylation. Each protocol makes use of two transient protection steps. In the case of tritylation of the 5' hydroxyl, the 2',3'-diol is protected by reaction with N,N-dimethylformamide dimethylacetal (Zemlicka, 1963) to prevent the small, but potentially troublesome, tritylation of the 2'-hydroxyl that otherwise accompanies tritylation of the 5'-hydroxyl (Zhang et al., 1997). The phenoxyacetylation of the amino group is carried out after transient hydroxyl and guanine O6 protection with trimethylchlorosilane using the hydroxybenzotriazole active ester of phenoxyacetic acid. These protocols give overall yields that are three times the best yields available by conventional procedures for adenosine and guanosine, but offer no advantage for cytidine or uridine. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-6-N-acyladenosine Basic Protocol 2: Synthesis of 5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethysilyl-2-N-acylguanosine.


Asunto(s)
Ribonucleósidos , Compuestos Organofosforados , ARN
14.
Cell Stem Cell ; 30(1): 3-4, 2023 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-36608677

RESUMEN

Chemical modifications of RNA are regulated by a series of readers, writers, and erasers that dictate gene expression. Two new studies in Cell Stem Cell1,2identify roles for the N6-methyladenosine (m6A) methyltransferase METTL16 and the m6A reader IGF2BP2 in leukemia-initiating cells, illuminating exciting new therapeutic targets for leukemia.


Asunto(s)
Leucemia , ARN , Humanos , ARN/genética , ARN/metabolismo , Metilación , Metiltransferasas/genética , Leucemia/genética , Células Madre/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
15.
Cells ; 12(1)2023 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-36611995

RESUMEN

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.


Asunto(s)
Carcinoma , Neoplasias del Colon , Humanos , ARN Mensajero/genética , Caspasa 7 , Ubiquitina-Proteína Ligasas/metabolismo , ARN , Neoplasias del Colon/genética , Línea Celular Tumoral , Ubiquitina , Apoptosis/genética , Proteínas de Motivos Tripartitos/genética , Factores de Transcripción/genética
16.
Bioinformatics ; 39(1)2023 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-36592044

RESUMEN

SUMMARY: Biological condensates are membraneless organelles with different material properties. Proteins and RNAs are the main components, but most of their interactions are still unknown. Here, we introduce PRALINE, a database for the interrogation of proteins and RNAs contained in stress granules, processing bodies and other assemblies including droplets and amyloids. PRALINE provides information about the predicted and experimentally validated protein-protein, protein-RNA and RNA-RNA interactions. For proteins, it reports the liquid-liquid phase separation and liquid-solid phase separation propensities. For RNAs, it provides information on predicted secondary structure content. PRALINE shows detailed information on human single-nucleotide variants, their clinical significance and presence in protein and RNA binding sites, and how they can affect condensates' physical properties. AVAILABILITY AND IMPLEMENTATION: PRALINE is freely accessible on the web at http://praline.tartaglialab.com.


Asunto(s)
Orgánulos , ARN , Humanos , ARN/metabolismo , Proteínas/metabolismo , Nucleótidos/metabolismo
17.
Nucleus ; 14(1): 2160551, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36602897

RESUMEN

Enhancers are cis-regulatory elements that can stimulate gene expression from distance, and drive precise spatiotemporal gene expression profiles during development. Functional enhancers display specific features including an open chromatin conformation, Histone H3 lysine 27 acetylation, Histone H3 lysine 4 mono-methylation enrichment, and enhancer RNAs production. These features are modified upon developmental cues which impacts their activity. In this review, we describe the current state of knowledge about enhancer functions and the diverse chromatin signatures found on enhancers. We also discuss the dynamic changes of enhancer chromatin signatures, and their impact on lineage specific gene expression profiles, during development or cellular differentiation.


Asunto(s)
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Lisina/metabolismo , Elementos de Facilitación Genéticos/genética , ARN
18.
BMC Neurosci ; 24(1): 1, 2023 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-36604634

RESUMEN

BACKGROUND: The role of cytokines in the pathophysiology, diagnosis, and prognosis of small fiber neuropathy (SFN) is incompletely understood. We studied expression profiles of selected pro- and anti-inflammatory cytokines in RNA from white blood cells (WBC) of patients with a medical history and a clinical phenotype suggestive for SFN and compared data with healthy controls. METHODS: We prospectively recruited 52 patients and 21 age- and sex-matched healthy controls. Study participants were characterized in detail and underwent complete neurological examination. Venous blood was drawn for routine and extended laboratory tests, and for WBC isolation. Systemic RNA expression profiles of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-2, IL-8, tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4, IL-10, transforming growth factor beta-1 (TGF) were analyzed. Protein levels of IL-2, IL-8, and TNF were measured in serum of patients and controls. Receiver operating characteristic (ROC)-curve analysis was used to determine the accuracy of IL-2, IL-8, and TNF in differentiating patients and controls. To compare the potential discriminatory efficacy of single versus combined cytokines, equality of different AUCs was tested. RESULTS: WBC gene expression of IL-2, IL-8, and TNF was higher in patients compared to healthy controls (IL-2: p = 0.02; IL-8: p = 0.009; TNF: p = 0.03) and discriminated between the groups (area under the curve (AUC) ≥ 0.68 for each cytokine) with highest diagnostic accuracy reached by combining the three cytokines (AUC = 0.81, sensitivity = 70%, specificity = 86%). Subgroup analysis revealed the following differences: IL-8 and TNF gene expression levels were higher in female patients compared to female controls (IL-8: p = 0.01; TNF: p = 0.03). The combination of TNF with IL-2 and TNF with IL-2 and IL-8 discriminated best between the study groups. IL-2 was higher expressed in patients with moderate pain compared to those with severe pain (p = 0.02). Patients with acral pain showed higher IL-10 gene expression compared to patients with generalized pain (p = 0.004). We further found a negative correlation between the relative gene expression of IL-2 and current pain intensity (p = 0.02). Serum protein levels of IL-2, IL-8, and TNF did not differ between patients and controls. CONCLUSIONS: We identified higher systemic gene expression of IL-2, IL-8, and TNF in SFN patients than in controls, which may be of potential relevance for diagnostics and patient stratification.


Asunto(s)
Citocinas , Neuropatía de Fibras Pequeñas , Femenino , Humanos , Interleucina-10 , Interleucina-2 , Interleucina-8 , Leucocitos/química , Dolor , ARN , Factor de Necrosis Tumoral alfa
19.
Sci Rep ; 13(1): 350, 2023 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-36611052

RESUMEN

In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein-protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues' interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14's exoribonuclease (ExoN) and nsp16's 2'O-methyltransferase (2'O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2'O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell's innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein-protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide-protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Simulación del Acoplamiento Molecular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Replicación Viral/genética , Metiltransferasas/genética , Péptidos/farmacología , Antivirales/farmacología , ARN/farmacología , Exorribonucleasas/genética , Exorribonucleasas/química
20.
PLoS One ; 18(1): e0280243, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36622844

RESUMEN

The importance of air purifiers has increased in recent years, especially with the "coronavirus disease 2019" pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.


Asunto(s)
Filtros de Aire , Azidas , Transcripción Reversa , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ARN , Sensibilidad y Especificidad , Técnicas de Amplificación de Ácido Nucleico/métodos
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