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1.
Nucleic Acids Res ; 51(12): 6227-6237, 2023 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-37207329

RESUMEN

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.


Asunto(s)
Fibrosis Pulmonar , ARN Largo no Codificante , Animales , Ratones , Fibrosis , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , ARN Largo no Codificante/metabolismo
2.
Chembiochem ; 25(10): e202400049, 2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38456652

RESUMEN

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.


Asunto(s)
ADN , Conformación de Ácido Nucleico , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , ADN/química , ADN/genética , Humanos , Ácidos Nucleicos Heterodúplex/química , ARN/química , ARN/genética , ARN/metabolismo , Termodinámica
3.
Brief Bioinform ; 23(6)2022 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-36239395

RESUMEN

RNA.DNA:DNA triple helix (triplex) formation is a form of RNA-DNA interaction which regulates gene expression but is difficult to study experimentally in vivo. This makes accurate computational prediction of such interactions highly important in the field of RNA research. Current predictive methods use canonical Hoogsteen base pairing rules, which whilst biophysically valid, may not reflect the plastic nature of cell biology. Here, we present the first optimization approach to learn a probabilistic model describing RNA-DNA interactions directly from motifs derived from triplex sequencing data. We find that there are several stable interaction codes, including Hoogsteen base pairing and novel RNA-DNA base pairings, which agree with in vitro measurements. We implemented these findings in TriplexAligner, a program that uses the determined interaction codes to predict triplex binding. TriplexAligner predicts RNA-DNA interactions identified in all-to-all sequencing data more accurately than all previously published tools in human and mouse and also predicts previously studied triplex interactions with known regulatory functions. We further validated a novel triplex interaction using biophysical experiments. Our work is an important step towards better understanding of triplex formation and allows genome-wide analyses of RNA-DNA interactions.


Asunto(s)
Estudio de Asociación del Genoma Completo , ARN , Humanos , Ratones , Animales , ARN/genética , ADN/genética , ADN/metabolismo , Replicación del ADN , Conformación de Ácido Nucleico
4.
Emerg Infect Dis ; 27(1)2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33261717

RESUMEN

We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted <25% false-positive results among controls, likely due to unspecific immune responses elicited by acute malaria. Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions.


Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Pruebas Serológicas , Benin , COVID-19/sangre , COVID-19/virología , Humanos , Sensibilidad y Especificidad
5.
Angew Chem Int Ed Engl ; 59(6): 2304-2308, 2020 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-31730253

RESUMEN

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in-cell NMR spectroscopy experiments. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.


Asunto(s)
Espectroscopía de Resonancia Magnética , Metabolómica/métodos , Línea Celular Tumoral , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Metaboloma/efectos de los fármacos , Estaurosporina/análogos & derivados , Estaurosporina/química , Estaurosporina/metabolismo , Estaurosporina/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo
6.
Front Immunol ; 15: 1305586, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38322252

RESUMEN

Introduction: One of the unexpected outcomes of the COVID-19 pandemic was the relatively low levels of morbidity and mortality in Africa compared to the rest of the world. Nigeria, Africa's most populous nation, accounted for less than 0.01% of the global COVID-19 fatalities. The factors responsible for Nigeria's relatively low loss of life due to COVID-19 are unknown. Also, the correlates of protective immunity to SARS-CoV-2 and the impact of pre-existing immunity on the outcome of the COVID-19 pandemic in Africa are yet to be elucidated. Here, we evaluated the natural and vaccine-induced immune responses from vaccinated, non-vaccinated and convalescent individuals in Southern Nigeria throughout the three waves of the COVID-19 pandemic in Nigeria. We also examined the pre-existing immune responses to SARS-CoV-2 from samples collected prior to the COVID-19 pandemic. Methods: We used spike RBD and N- IgG antibody ELISA to measure binding antibody responses, SARS-CoV-2 pseudotype assay protocol expressing the spike protein of different variants (D614G, Delta, Beta, Omicron BA1) to measure neutralizing antibody responses and nucleoprotein (N) and spike (S1, S2) direct ex vivo interferon gamma (IFNγ) T cell ELISpot to measure T cell responses. Result: Our study demonstrated a similar magnitude of both binding (N-IgG (74% and 62%), S-RBD IgG (70% and 53%) and neutralizing (D614G (49% and 29%), Delta (56% and 47%), Beta (48% and 24%), Omicron BA1 (41% and 21%)) antibody responses from symptomatic and asymptomatic survivors in Nigeria. A similar magnitude was also seen among vaccinated participants. Interestingly, we revealed the presence of preexisting binding antibodies (N-IgG (60%) and S-RBD IgG (44%)) but no neutralizing antibodies from samples collected prior to the pandemic. Discussion: These findings revealed that both vaccinated, non-vaccinated and convalescent individuals in Southern Nigeria make similar magnitude of both binding and cross-reactive neutralizing antibody responses. It supported the presence of preexisting binding antibody responses among some Nigerians prior to the COVID-19 pandemic. Lastly, hybrid immunity and heterologous vaccine boosting induced the strongest binding and broadly neutralizing antibody responses compared to vaccine or infection-acquired immunity alone.


Asunto(s)
COVID-19 , Pueblo de África Occidental , Humanos , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , COVID-19/inmunología , Ensayo de Immunospot Ligado a Enzimas , Inmunoglobulina G , Nigeria , Pandemias , SARS-CoV-2
7.
Nat Commun ; 13(1): 6563, 2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36323673

RESUMEN

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.


Asunto(s)
ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Endoteliales/metabolismo , ADN/genética , ADN/metabolismo , Emparejamiento Base , Oligonucleótidos , Regulación Neoplásica de la Expresión Génica
8.
Sci Rep ; 12(1): 22330, 2022 12 25.
Artículo en Inglés | MEDLINE | ID: mdl-36567369

RESUMEN

Elucidating the adaptive immune characteristics of natural protection to Lassa fever (LF) is vital in designing and selecting optimal vaccine candidates. With rejuvenated interest in LF and a call for accelerated research on the Lassa virus (LASV) vaccine, there is a need to define the correlates of natural protective immune responses to LF. Here, we describe cellular and antibody immune responses present in survivors of LF (N = 370) and their exposed contacts (N = 170) in a LASV endemic region in Nigeria. Interestingly, our data showed comparable T cell and binding antibody responses from both survivors and their contacts, while neutralizing antibody responses were primarily seen in the LF survivors and not their contacts. Neutralizing antibody responses were found to be cross-reactive against all five lineages of LASV with a strong bias to Lineage II, the prevalent strain in southern Nigeria. We demonstrated that both T cell and antibody responses were not detectable in peripheral blood after a decade in LF survivors. Notably LF survivors maintained high levels of detectable binding antibody response for six months while their contacts did not. Lastly, as potential vaccine targets, we identified the regions of the LASV Glycoprotein (GP) and Nucleoprotein (NP) that induced the broadest peptide-specific T cell responses. Taken together this data informs immunological readouts and potential benchmarks for clinical trials evaluating LASV vaccine candidates.


Asunto(s)
Fiebre de Lassa , Virus Lassa , Humanos , Nigeria/epidemiología , Inmunidad Celular , Anticuerpos Neutralizantes , Sobrevivientes
9.
mSphere ; 6(1)2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33441410

RESUMEN

Information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Africa is limited by insufficient diagnostic capacity. Here, we assessed the coronavirus disease (COVID-19)-related diagnostic workload during the onset of the pandemic in the central laboratory of Benin, Western Africa; characterized 12 SARS-CoV-2 genomes from returning travelers; and validated the Da An RT-PCR-based diagnostic kit that is widely used across Africa. We found a 15-fold increase in the monthly laboratory workload due to COVID-19, dealt with at the cost of routine activities. Genomic surveillance showed near-simultaneous introduction of distinct SARS-CoV-2 lineages termed A.4 and B.1, including the D614G spike protein variant potentially associated with higher transmissibility from travelers from six different European and African countries during March-April 2020. We decoded the target regions within the ORF1ab and N genes of the Da An dual-target kit by MinION-based amplicon sequencing. Despite relatively high similarity between SARS-CoV-2 and endemic human coronaviruses (HCoVs) within the ORF1ab target domain, no cross-detection of high-titered cell culture supernatants of HCoVs was observed, suggesting high analytical specificity. The Da An kit was highly sensitive, detecting 3.2 to 9.0 copies of target-specific in vitro transcripts/reaction. Although discrepant test results were observed in low-titered clinical samples, clinical sensitivity of the Da An kit was at least comparable to that of commercial kits from affluent settings. In sum, virologic diagnostics are achievable in a resource-limited setting, but unprecedented pressure resulting from COVID-19-related diagnostics requires rapid and sustainable support of national and supranational stakeholders addressing limited laboratory capacity.IMPORTANCE Months after the start of the COVID-19 pandemic, case numbers from Africa are surprisingly low, potentially because the number of SARS-CoV-2 tests performed in Africa is lower than in other regions. Here, we show an overload of COVID-19-related diagnostics in the central laboratory of Benin, Western Africa, with a stagnating average number of positive samples irrespective of daily sample counts. SARS-CoV-2 genomic surveillance confirmed a high genomic diversity in Benin introduced by travelers returning from Europe and other African countries, including early circulation of the D614G spike mutation associated with potentially higher transmissibility. We validated a widely used RT-PCR kit donated by the Chinese Jack Ma Foundation and confirmed high analytical specificity and clinical sensitivity equivalent to tests used in affluent settings. Our assessment shows that although achievable in an African setting, the burden from COVID-19-related diagnostics on national reference laboratories is very high.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Adulto , Benin/epidemiología , COVID-19/epidemiología , COVID-19/transmisión , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/métodos , Prueba de Ácido Nucleico para COVID-19/estadística & datos numéricos , Países en Desarrollo , Femenino , Genoma Viral , Recursos en Salud/provisión & distribución , Accesibilidad a los Servicios de Salud/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/genética , Sensibilidad y Especificidad , Enfermedad Relacionada con los Viajes , Carga de Trabajo/estadística & datos numéricos
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