RESUMEN
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.
Asunto(s)
Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/virología , Endosomas/virología , Interacciones Huésped-Patógeno/fisiología , Proteínas Virales/metabolismo , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Chlorocebus aethiops , Endosomas/metabolismo , Células HEK293 , Humanos , Porcinos , Células VeroRESUMEN
OBJECTIVE: To evaluate cell-free DNA (cfDNA) testing as a non-invasive approach to detecting aneuploidies in clinical miscarriages. DESIGN: A retrospective cohort study of women with pregnancy loss. SETTING: Hospitals and genetic analysis laboratories. POPULATION OR SAMPLE: Pregnancy losses in the period 2021-2022. METHODS: Results derived from non-invasive cfDNA testing (Veriseq NIPT Solution V2) of maternal blood and invasive analysis of products of conception (POC) (Ion ReproSeq) compared in 120 women who suffered a miscarriage. MAIN OUTCOME MEASURES: Concordance rate results, cfDNA testing performance, non-informative rate (NIR) and fetal fraction (FF). RESULTS: We found no significant differences in the NIR between invasive (iPOC) and non-invasive (niPOC) analysis of POC (10.0% [12/120] versus 16.7% [20/120]). Of 120 samples, 90 provided an informative result in iPOC and niPOC groups (75%). cfDNA analysis correctly identified 74/87 (85.1%) samples (excluding triploidies). Sensitivity and specificity were 79.4% and 100%, respectively; all discordant cases were female. A binomial logistic model suggested fetal sex as the only variable influencing the concordance rate (P = 0.035). A Y-chromosome-based FF estimate allowed the optimal reclassification of cfDNA of non-informative male fetuses and a more accurate evaluation of cfDNA testing performance. The difference between the two FF estimates (native algorithm and Y-chromosome-based) suggests that female non-concordant cases may represent non-informative cases. CONCLUSIONS: Cell-free DNA-based testing provides a non-invasive approach to determining the genetic cause of clinical miscarriage.
Asunto(s)
Aborto Espontáneo , Ácidos Nucleicos Libres de Células , Embarazo , Femenino , Masculino , Humanos , Aborto Espontáneo/diagnóstico , Aborto Espontáneo/genética , Estudios Retrospectivos , Diagnóstico Prenatal/métodos , Aneuploidia , TrisomíaRESUMEN
Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Mebendazol , Animales , Antivirales/farmacología , Mamíferos , Mebendazol/farmacología , Microtúbulos , SARS-CoV-2 , Tubulina (Proteína)RESUMEN
We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid embryos. In contrast, the number of MII oocytes retrieved or the use of oocyte vitrification did not affect aneuploidy rates. Similarly, neither maternal age, oocyte number, nor oocyte vitrification affected the incidence of mosaicism. Analysis of clinical outcomes, indications, and potential benefits of embryo aneuploidy testing revealed advanced maternal age as the most favored group, with some evidence of improved delivery rate per transfer as well as decreased miscarriage rates and time to pregnancy. Other indications are: recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and good prognosis patients mainly undergoing single embryo transfer, with the latter indication used to reduce the occurrence of multiple pregnancies without compromising cycle outcome. In conclusion, NGS has become the most appropriate technology for aneuploidy testing in trophectoderm biopsies, with accurate results, high throughput, and cost efficiency. This technology can be also applied to the analysis of the embryonic cell free DNA released to the culture media at blastocyst stage. This is a promising approach towards a non-invasive preimplantation genetic testing of aneuploidy.
Asunto(s)
Aneuploidia , Análisis Citogenético/métodos , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Diagnóstico Preimplantación/métodos , Blastocisto/química , Blastocisto/citología , Ácidos Nucleicos Libres de Células/análisis , Ácidos Nucleicos Libres de Células/genética , Análisis Citogenético/tendencias , Transferencia de Embrión , Femenino , Pruebas Genéticas/tendencias , Humanos , Masculino , Mosaicismo , Pruebas Prenatales no Invasivas/métodos , Pruebas Prenatales no Invasivas/tendencias , Medicina de Precisión , Embarazo , Diagnóstico Preimplantación/tendencias , Factores de Riesgo , Factores de TiempoRESUMEN
In this work we reviewed 18 years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.
Asunto(s)
Aneuploidia , Aberraciones Cromosómicas/embriología , Diagnóstico Preimplantación , Espermatozoides/anomalías , Hibridación Genómica Comparativa , Femenino , Fertilización In Vitro , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Infertilidad Masculina/genética , Infertilidad Masculina/terapia , Masculino , Oligospermia/genética , Medicina de Precisión , Embarazo , Estudios Retrospectivos , Recuento de Espermatozoides , Inyecciones de Esperma Intracitoplasmáticas , Motilidad Espermática , Espermatozoides/ultraestructuraRESUMEN
Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.
Asunto(s)
Virus de la Fiebre Porcina Africana/efectos de los fármacos , Antivirales/farmacología , Perileno/análogos & derivados , Uracilo/análogos & derivados , Uridina/análogos & derivados , Proteínas Virales/antagonistas & inhibidores , Virión/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Antivirales/síntesis química , Membrana Celular/efectos de los fármacos , Membrana Celular/virología , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Pruebas de Sensibilidad Microbiana , Perileno/síntesis química , Perileno/farmacología , Cultivo Primario de Células , Porcinos , Uracilo/síntesis química , Uracilo/farmacología , Uridina/síntesis química , Uridina/farmacología , Células Vero , Proteínas Virales/biosíntesis , Virión/crecimiento & desarrollo , Virión/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. RESULTS: Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. CONCLUSIONS: The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Dineínas/metabolismo , Nanopartículas del Metal/química , Nanotecnología/métodos , Secuencia de Aminoácidos , Animales , Línea Celular , Oro/química , Humanos , Nanopartículas del Metal/ultraestructura , Microtúbulos/metabolismo , Peso Molecular , Membrana Nuclear/metabolismo , Péptidos/química , Péptidos/metabolismo , Unión ProteicaRESUMEN
UNLABELLED: African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. IMPORTANCE: Since its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Colesterol/metabolismo , Endosomas/metabolismo , Endosomas/virología , Internalización del Virus , Animales , Chlorocebus aethiops , Concentración de Iones de Hidrógeno , Análisis de Flujos Metabólicos , Células VeroRESUMEN
PURPOSE: To analyze the molecular cytogenetic data obtained from products of conception (POC) obtained by selective biopsy of first trimester miscarriages and to estimate the rate of chromosomal anomalies in miscarriages from pregnancies achieved by natural conception (NC) or by assisted reproductive technology (ART) interventions. METHODS: We used KaryoLite™ BoBs™ (PerkinElmer LAS, Wallac, Turku, Finland) technology to analyze 189 samples from ART or NC pregnancies. RESULTS: All POC were successfully evaluated. A higher incidence of chromosomal abnormalities was observed in POC after ART using the patient's own oocytes than from NC pregnancies (62.7% vs. 40.6%; p < 0.05). The lowest incidence of chromosomal abnormalities was observed in POCs ART using donor eggs from women younger than 35 years (12.8%). No statistical differences in the percentage of abnormal miscarriages were observed in correlation with sperm concentration: a sperm concentration less than 5 million/mL produced 75% abnormal results and a concentration higher than 5 million/mL produced 51%. CONCLUSIONS: POC analysis is essential to determine the cause of pregnancy loss. Using culture-independent molecular biology techniques to analyze POCs avoids limitations such as growth failure and reduces the time required for analysis. Selective biopsy of fetal tissue by hysteroembryoscopy avoids the risk of misdiagnosis due to maternal cell contamination. Our results show that maternal age, sperm quality, and ART-assisted pregnancies are risk factors for abnormal gestations.
Asunto(s)
Aborto Espontáneo/patología , Aberraciones Cromosómicas , Fertilización/genética , Fetoscopía/métodos , Histeroscopía/métodos , Infertilidad/terapia , Resultado del Embarazo , Técnicas Reproductivas Asistidas/efectos adversos , Aborto Espontáneo/etiología , Adulto , Análisis Citogenético , Femenino , Humanos , Masculino , Edad Materna , Embarazo , Primer Trimestre del Embarazo/genética , Recuento de EspermatozoidesRESUMEN
The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection.
Asunto(s)
Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/metabolismo , Colesterol/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Chlorocebus aethiops , Porcinos , Células VeroRESUMEN
The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca2+-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.
Asunto(s)
Virus de la Fiebre Porcina Africana , Complejos de Clasificación Endosomal Requeridos para el Transporte , Porcinos , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Virus de la Fiebre Porcina Africana/genética , Proteínas de Unión al Calcio/metabolismo , Endosomas/metabolismo , EndocitosisRESUMEN
African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/fisiología , Proteínas Virales de Fusión/metabolismo , Proteómica , Línea CelularRESUMEN
Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Simulación de Dinámica Molecular , Mutagénesis , Replicación ViralRESUMEN
African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKß. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme.
Asunto(s)
Virus de la Fiebre Porcina Africana/enzimología , Inmunidad Innata , Inmunomodulación , FN-kappa B/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/inmunología , Células A549 , Virus de la Fiebre Porcina Africana/inmunología , Animales , Células HEK293 , Humanos , Interferón Tipo I/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Porcinos , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19.
Asunto(s)
Transporte Biológico , Tratamiento Farmacológico de COVID-19 , Endosomas/virología , Proteína Niemann-Pick C1/análisis , SARS-CoV-2/fisiología , Animales , Carbazoles/farmacología , Chlorocebus aethiops , Endosomas/química , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fusión de Membrana , Células Vero , Replicación ViralRESUMEN
Despite the efforts to develop new treatments against Ebola virus (EBOV) there is currently no antiviral drug licensed to treat patients with Ebola virus disease (EVD). Therefore, there is still an urgent need to find new drugs to fight against EBOV. In order to do this, a virtual screening was done on the druggable interaction between the EBOV glycoprotein (GP) and the host receptor NPC1 with a subsequent selection of compounds for further validation. This screening led to the identification of new small organic molecules with potent inhibitory action against EBOV infection using lentiviral EBOV-GP-pseudotype viruses. Moreover, some of these compounds have shown their ability to interfere with the intracellular cholesterol transport receptor NPC1 using an ELISA-based assay. These preliminary results pave the way to hit to lead optimization programs that lead to successful candidates.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/métodos , Proteína Niemann-Pick C1/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Chlorocebus aethiops , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Humanos , Células VeroRESUMEN
BACKGROUND: Since 2011, screening maternal blood for cell-free foetal DNA (cffDNA) fragments has offered a robust clinical tool to classify pregnancy as low or high-risk for Down, Edwards, and Patau syndromes. With recent advances in molecular biology and improvements in data analysis algorithms, the screening's scope of analysis continues to expand. Indeed, screening now encompassess additional conditions, including aneuploidies for sex chromosomes, microdeletions and microduplications, rare autosomal trisomies, and, more recently, segmental deletions and duplications called copy number variations (CNVs). Yet, the ability to detect CNVs creates a new challenge for cffDNA analysis in couples in which one member carries a structural rearrangement such as a translocation or inversion. CASE PRESENTATION: We report a segmental duplication of the long arm of chromosome 3 and a segmental deletion of the short arm of chromosome 5 detected by cffDNA analysis in a 25-year-old pregnant woman. The blood sample was sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. G-band karyotyping in amniotic fluid only detected an abnormality in chromosome 5. Next-generation sequencing in amniocytes confirmed both abnormalities and identified breakpoints in 3q26.32q29 and 5p13.3p15. The foetus died at 21 weeks of gestation due to multiple abnormalities, and later G-band karyotyping in the parents revealed that the father was a carrier of a balanced reciprocal translocation [46,XY,t(3;5)(q26.2;p13)]. Maternal karyotype appeared normal. CONCLUSION: This case provides evidence that extended cffDNA can detect, in addition to aneuploidies for whole chromosomes, large segmental aneuploidies. In some cases, this may indicate the presence of chromosomal rearrangements in a parent. Such abnormalities are outside the scope of standard cffDNA analysis targeting chromosomes 13, 18, 21, X, and Y, potentially leading to undiagnosed congenital conditions.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Cromosomas Humanos Par 3/genética , Enfermedades Fetales/genética , Feto/metabolismo , Trisomía/genética , Adulto , Biomarcadores/sangre , Cromosomas Humanos Par 3/metabolismo , Variaciones en el Número de Copia de ADN , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/metabolismo , Pruebas Genéticas , Humanos , Cariotipificación , Embarazo , Trisomía/diagnósticoRESUMEN
Highly sensitive next-generation sequencing (NGS) platforms applied to preimplantation genetic testing for aneuploidy (PGT-A) allow the classification of mosaicism in trophectoderm biopsies. However, the incidence of mosaicism reported by these tests can be affected by a wide number of analytical, biological, and clinical factors. With the use of a proprietary algorithm for automated diagnosis of aneuploidy and mosaicism, we retrospectively analyzed a large series of 115,368 trophectoderm biopsies from 27,436 PGT-A cycles to determine whether certain biological factors and in vitro fertilization (IVF) practices influence the incidence of overall aneuploidy, whole uniform aneuploidy, mosaicism, and TE biopsies with only segmental aneuploidy. Older female and male patients showed higher rates of high-mosaic degree and whole uniform aneuploidies and severe oligozoospermic patients had higher rates of mosaicism and only segmental aneuploidies. Logistic regression analysis identified a positive effect of female age but a negative effect of embryo vitrification on the incidence of overall aneuploid embryos. Female age increased whole uniform aneuploidy rates but decreased only segmental aneuploidy and mosaicism, mainly low-mosaics. Conversely, higher ovarian response decreased whole uniform aneuploidy rates but increased only segmental aneuploidies. Finally, embryo vitrification decreased whole uniform aneuploidy rates but increased mosaicism, mainly low-mosaics, compared to PGT-A cycles with fresh oocytes. These results could be useful for clinician's management of the IVF cycles.
Asunto(s)
Aneuploidia , Fertilización In Vitro , Pruebas Genéticas , Mosaicismo , Diagnóstico Preimplantación , Adulto , Biopsia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Incidencia , Masculino , Edad Materna , Edad Paterna , Estudios Retrospectivos , Recuento de Espermatozoides , TrofoblastosRESUMEN
African Swine Fever virus (ASFV) causes one of the most relevant emerging diseases affecting swine, now extended through three continents. The virus has a large coding capacity to deploy an arsenal of molecules antagonizing the host functions. In the present work, we have studied the only known E2 viral-conjugating enzyme, UBCv1 that is encoded by the I215L gene of ASFV. UBCv1 was expressed as an early expression protein that accumulates throughout the course of infection. This versatile protein, bound several types of polyubiquitin chains and its catalytic domain was required for enzymatic activity. High throughput mass spectrometry analysis in combination with a screening of an alveolar macrophage library was used to identify and characterize novel UBCv1-host interactors. The analysis revealed interaction with the 40S ribosomal protein RPS23, the cap-dependent translation machinery initiation factor eIF4E, and the E3 ubiquitin ligase Cullin 4B. Our data show that during ASFV infection, UBCv1 was able to bind to eIF4E, independent from the cap-dependent complex. Our results provide novel insights into the function of the viral UBCv1 in hijacking cellular components that impact the mTORC signaling pathway, the regulation of the host translation machinery, and the cellular protein expression during the ASFV lifecycle.
RESUMEN
Background: An antibody panel is needed to definitively differentiate between adenocarcinoma (AC) and squamous cell carcinoma (SCC) in order to meet more stringent requirements for the histologic classification of lung cancers. Staining of desmosomal plaque-related proteins may be useful in the diagnosis of lung SCC.Materials and methods: We compared the usefulness of six conventional (CK5/6, p40, p63, CK7, TTF1, and Napsin A) and three novel (PKP1, KRT15, and DSG3) markers to distinguish between lung SCC and AC in 85 small biopsy specimens (41 ACs and 44 SCCs). Correlations were examined between expression of the markers and patients' histologic and clinical data.Results: The specificity for SCC of membrane staining for PKP1, KRT15, and DSG3 was 97.4%, 94.6%, and 100%, respectively, and it was 100% when the markers were used together and in combination with the conventional markers (AUCs of 0.7619 for Panel 1 SCC, 0.7375 for Panel 2 SCC, 0.8552 for Panel 1 AC, and 0.8088 for Panel 2 AC). In a stepwise multivariate logistic regression model, the combination of CK5/6, p63, and PKP1 in membrane was the optimal panel to differentiate between SCC and AC, with a percentage correct classification of 96.2% overall (94.6% of ACs and 97.6% of SCCs). PKP1 and DSG3 are related to the prognosis.Conclusions: PKP1, KRT15, and DSG3 are highly specific for SCC, but they were more useful to differentiate between SCC and AC when used together and in combination with conventional markers. PKP1 and DSG3 expressions may have prognostic value.