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Solar particle events (SPEs) are short-lived bursts of high-energy particles from the solar atmosphere and are widely recognized as posing significant economic risks to modern society. Most SPEs are relatively weak and have minor impacts on the Earth's environment, but historic records contain much stronger SPEs which have the potential to alter atmospheric chemistry, impacting climate and biological life. The impacts of such strong SPEs would be far more severe when the Earth's protective geomagnetic field is weak, such as during past geomagnetic excursions or reversals. Here, we model the impacts of an extreme SPE under different geomagnetic field strengths, focusing on changes in atmospheric chemistry and surface radiation using the atmosphere-ocean-chemistry-climate model SOCOL3-MPIOM and the radiation transfer model LibRadtran. Under current geomagnetic conditions, an extreme SPE would increase NOx concentrations in the polar stratosphere and mesosphere, causing reductions in extratropical stratospheric ozone lasting for about a year. In contrast, with no geomagnetic field, there would be a substantial increase in NOx throughout the entire atmosphere, resulting in severe stratospheric ozone depletion for several years. The resulting ground-level ultraviolet (UV) radiation would remain elevated for up to 6 y, leading to increases in UV index up to 20 to 25% and solar-induced DNA damage rates by 40 to 50%. The potential evolutionary impacts of past extreme SPEs remain an important question, while the risks they pose to human health in modern conditions continue to be underestimated.
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The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.
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Poli A , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Poli A/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Células HeLa , Caperuzas de ARN/metabolismo , Estabilidad del ARNRESUMEN
Polyelectrolyte complexes (PECs) based on polysaccharides, including hyaluronic acid (HA) and chitosan (CS), are promising delivery systems for antimicrobial agents, including oral administration of the peptide antibiotic colistin (CT). Modification of CS with different targeting ligands to improve intestinal permeability is a suitable way to improve the oral bioavailability of polyelectrolyte particles. This study describes the procedure for obtaining CT-containing PECs based on HA and CS modified with cyanocobalamin (vitamin B12). In this case, vitamin B12 is used as a targeting ligand because it is absorbed in the ileum via specific transporter proteins. The resulting PECs had a hydrodynamic size of about 284 nm and a positive ζ-potential of about 26 mV; the encapsulation efficiency was 88.2 % and the CT content was 42.2 µg/mg. The developed systems provided a two-phase drug release: about 50 % of the CT was released in 0.5-1 h, and about 60 % of the antibiotic was cumulatively released in 5 h. The antimicrobial activity of encapsulated CT was maintained at the same level as the pure drug for at least 24 h (minimum inhibitory concentration against Pseudomonas aeruginosa was 2 µg/mL for both). In addition, the apparent permeability coefficient of CT in the PEC formulation was 2.4 × 10-6 cm/s. Thus, the incorporation of CT into HA- and vitamin B12-modified CS-based PECs can be considered as a simple and convenient method to improve the oral delivery of CT.
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Quitosano , Polielectrolitos/química , Quitosano/química , Portadores de Fármacos/química , Ácido Hialurónico , Colistina/farmacología , Vitamina B 12 , Administración Oral , Antibacterianos/farmacologíaRESUMEN
RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the selective suppression of the heterozygous GNAO1 c.607 G > A variant causing GNAO1 encephalopathy. By screening short interfering RNA (siRNA), we showed that GNAO1 c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant GNAO1, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic GNAO1 c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.
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Encefalopatías , Terapia Genética , Animales , Humanos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Alelos , Encefalopatías/genética , Vectores Genéticos/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genéticaRESUMEN
Annual bone grafting surgeries due to bone fractures, resections of affected bones, skeletal anomalies, osteoporosis, etc. exceed two million worldwide. In this regard, the creation of new materials for bone tissue repair is one of the urgent tasks of modern medicine. Additive manufacturing, or 3D printing, offers great opportunities for the development of materials with diverse properties and designs. In this study, the one-pot technique for the production of 3D scaffolds based on poly(ε-caprolactone) (PCL) loaded with an antibiotic or anti-inflammatory drug was proposed. In contrast to previously described methods to prepare drug-containing scaffolds, drug-loaded PCL scaffolds were prepared by direct 3D printing from a polymer/drug blend. An investigation of the mechanical properties of 3D-printed scaffolds containing 0.5-5 wt% ciprofloxacin (CIP) or dexamethasone (DEX) showed almost no effect of the drug (compression modulus ~70-90 MPa) compared to unfilled PCL (74 MPa). At the same time, introducing the drug and increasing its content in the PCL matrix contributed to a 1.8-6.8-fold decrease in the specific surface area of the scaffold, depending on composition. The release of CIP and DEX in phosphate buffer solution and in the same buffer containing lipase revealed a faster release in enzyme-containing medium within 45 days. Furthermore, drug release was more intensive from scaffolds with a low drug load. Analysis of the release profiles using a number of mathematical dissolution models led to the conclusion that diffusion dominates over other probable factors. In vitro biological evaluation of the scaffolds containing DEX showed moderate toxicity against osteoblast-like and leukemia monocytic cells. Being 3D-printed together with PCL both drugs retain their biological activity. PCL/CIP and PCL/DEX scaffolds demonstrated antibacterial properties against Pseudomonas aeruginosa (a total inhibition after 48 h) and anti-inflammatory activity in experiments on TNFα-activated monocyte cells (a 4-time reduction in CD-54 expression relative to control), respectively.
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Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a deficiency in the dystrophin protein. The most frequent types of disease-causing mutations in the DMD gene are frameshift deletions of one or more exons. Precision genome editing systems such as CRISPR-Cas9 have shown potential to restore open reading frames in numerous animal studies. Here, we applied an AAV-CRISPR double-cut strategy to correct a mutation in the DMD mouse model with exon 8-34 deletion, encompassing the N-terminal actin-binding domain. We report successful excision of the 100-kb genomic sequence, which includes exons 6 and 7, and partial improvement in cardiorespiratory function. While corrected mRNA was abundant in muscle tissues, only a low level of truncated dystrophin was produced, possibly because of protein instability. Furthermore, CRISPR-Cas9-mediated genome editing upregulated the Dp71f dystrophin isoform on the sarcolemma. Given the previously reported Dp71-associated muscle pathology, our results question the applicability of genome editing strategies for some DMD patients with N-terminal mutations. The safety and efficacy of CRISPR-Cas9 constructs require rigorous investigation in patient-specific animal models.
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Polymeric drug delivery systems enhance the biopharmaceutical properties of antibiotics by increasing their bioavailability, providing programmable and controlled-release properties, and reducing toxicity. In addition, drug delivery systems are a promising strategy to improve the intestinal permeability of various antimicrobial agents, including colistin (CT). This study describes the modification of conjugates based on CT and hyaluronic acid (HA) with cyanocobalamin (vitamin B12). Vitamin B12 was chosen as a targeting ligand because it has its own absorption pathway in the small intestine. The resulting polysaccharide conjugates contained 95 µg/mg vitamin B12 and the CT content was 335 µg/mg; they consisted of particles of two sizes, 98 and 702 nm, with a ζ-potential of approximately -25 mV. An in vitro release test at pH 7.4 and pH 5.2 showed an ultra-slow release of colistin of approximately 1% after 10 h. The modified B12 conjugates retained their antimicrobial activity at the level of pure CT (minimum inhibitory concentration was 2 µg/mL). The resulting delivery systems also reduced the nephrotoxicity of CT by 30-40% (HEK 293 cell line). In addition, the modification of B12 improved the intestinal permeability of CT, and the apparent permeability coefficient of HA-CT-B12 conjugates was 3.5 × 10-6 cm/s, corresponding to an in vivo intestinal absorption of 50-100%. Thus, vitamin-B12-modified conjugates based on CT and HA may be promising oral delivery systems with improved biopharmaceutical properties.
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Colistina , Ácido Hialurónico , Humanos , Colistina/farmacología , Ácido Hialurónico/química , Células HEK293 , Vitamina B 12 , Sistemas de Liberación de Medicamentos/métodosRESUMEN
Frontiers in theranostics are driving the demand for multifunctional nanoagents. Upconversion nanoparticle (UCNP)-based systems activated by near-infrared (NIR) light deeply penetrating biotissue are a powerful tool for the simultaneous diagnosis and therapy of cancer. The intercalation into large polymer micelles of poly(maleic anhydride-alt-1-octadecene) provided the creation of biocompatible UCNPs. The intrinsic properties of UCNPs (core@shell structure NaYF4:Yb3+/Tm3+@NaYF4) embedded in micelles delivered NIR-to-NIR visualization, photothermal therapy, and high drug capacity. Further surface modification of micelles with a thermosensitive polymer (poly-N-vinylcaprolactam) exhibiting a conformation transition provided gradual drug (doxorubicin) release. In addition, the decoration of UCNP micelles with Ag nanoparticles (Ag NPs) synthesized in situ by silver ion reduction enhanced the cytotoxicity of micelles at cell growth temperature. Cell viability assessment on Sk-Br-3, MDA-MB-231, and WI-26 cell lines confirmed this effect. The efficiency of the prepared UCNP complex was evaluated in vivo by Sk-Br-3 xenograft regression in mice for 25 days after peritumoral injection and photoactivation of the lesions with NIR light. The designed polymer micelles hold promise as a photoactivated theranostic agent with quattro-functionalities (NIR absorption, photothermal effect, Ag NP cytotoxicity, and Dox loading) that provides imaging along with chemo- and photothermal therapy enhanced with Ag NPs.
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Nanopartículas del Metal , Nanopartículas , Neoplasias , Humanos , Animales , Ratones , Micelas , Terapia Fototérmica , Plata , Nanopartículas/química , Polímeros/química , Doxorrubicina/química , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Línea Celular TumoralRESUMEN
Water vapor plays an important role in many aspects of the climate system, by affecting radiation, cloud formation, atmospheric chemistry and dynamics. Even the low stratospheric water vapor content provides an important climate feedback, but current climate models show a substantial moist bias in the lowermost stratosphere. Here we report crucial sensitivity of the atmospheric circulation in the stratosphere and troposphere to the abundance of water vapor in the lowermost stratosphere. We show from a mechanistic climate model experiment and inter-model variability that lowermost stratospheric water vapor decreases local temperatures, and thereby causes an upward and poleward shift of subtropical jets, a strengthening of the stratospheric circulation, a poleward shift of the tropospheric eddy-driven jet and regional climate impacts. The mechanistic model experiment in combination with atmospheric observations further shows that the prevailing moist bias in current models is likely caused by the transport scheme, and can be alleviated by employing a less diffusive Lagrangian scheme. The related effects on atmospheric circulation are of similar magnitude as climate change effects. Hence, lowermost stratospheric water vapor exerts a first order effect on atmospheric circulation and improving its representation in models offers promising prospects for future research.
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Mutations that prevent the production of proteins in the DMD gene cause Duchenne muscular dystrophy. Most frequently, these are deletions leading to reading-frame shift. The "reading-frame rule" states that deletions that preserve ORF result in a milder Becker muscular dystrophy. By removing several exons, new genome editing tools enable reading-frame restoration in DMD with the production of BMD-like dystrophins. However, not every truncated dystrophin with a significant internal loss functions properly. To determine the effectiveness of potential genome editing, each variant should be carefully studied in vitro or in vivo. In this study, we focused on the deletion of exons 8-50 as a potential reading-frame restoration option. Using the CRISPR-Cas9 tool, we created the novel mouse model DMDdel8-50, which has an in-frame deletion in the DMD gene. We compared DMDdel8-50 mice to C57Bl6/CBA background control mice and previously generated DMDdel8-34 KO mice. We discovered that the shortened protein was expressed and correctly localized on the sarcolemma. The truncated protein, on the other hand, was unable to function like a full-length dystrophin and prevent disease progression. On the basis of protein expression, histological examination, and physical assessment of the mice, we concluded that the deletion of exons 8-50 is an exception to the reading-frame rule.
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Distrofina , Distrofia Muscular de Duchenne , Ratones , Animales , Distrofina/genética , Ratones Endogámicos CBA , Distrofia Muscular de Duchenne/metabolismo , Fenotipo , Exones/genética , Eliminación de GenRESUMEN
The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the "humanized" fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.
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The emergence and growth of bacterial resistance to antibiotics poses an enormous threat to humanity in the future. In this regard, the discovery of new antibiotics and the improvement of existing ones is a priority task. In this study, we proposed the synthesis of new polymeric conjugates of polymyxin B, which is a clinically approved but limited-use peptide antibiotic. In particular, three carboxylate-bearing polymers and one synthetic glycopolymer were selected for conjugation with polymyxin B (PMX B), namely, poly(α,L-glutamic acid) (PGlu), copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)), copolymer of N-vinyl succinamic acid and N-vinylsuccinimide (P(VSAA-co-VSI)), and poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG). Unlike PGlu and PMAG, P(Glu-co-Phe) and P(VSAA-co-VSI) are amphiphilic and form nanoparticles in aqueous media. A number of conjugates with different polymyxin B loading were synthesized and characterized. In addition, the complex conjugates of PGLu or PMAG with polymyxin B and deferoxamine (siderophore) were obtained. A release of PMX B from Schiff base and amide-linked polymer conjugates was studied in model buffer media with pH 7.4 and 5.8. In both cases, a more pronounced release was observed under slightly acidic conditions. The cytotoxicity of free polymers and PMX B as well as their conjugates was examined in human embryonic kidney cells (HEK 293T cell line). All conjugates demonstrated reduced cytotoxicity compared to the free antibiotic. Finally, the antimicrobial efficacy of the conjugates against Pseudomonas aeruginosa was determined and compared. The lowest values of minimum inhibitory concentrations (MIC) were observed for polymyxin B and polymyxin B/deferoxamine conjugated with PMAG. Among the polymers tested, PMAG appears to be the most promising carrier for delivery of PMX B in conjugated form due to the good preservation of the antimicrobial properties of PMX B and the ability of controlled drug release.
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Deferoxamina , Polimixina B , Humanos , Polimixina B/farmacología , Ácido Glutámico , Antibacterianos/farmacología , Polímeros/químicaRESUMEN
Mitochondrial diseases (MD) are a heterogeneous group of multisystem disorders involving metabolic errors. MD are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystem dysfunction with different clinical courses. Most primary MD are autosomal recessive but maternal inheritance (from mtDNA), autosomal dominant, and X-linked inheritance is also known. Mitochondria are unique energy-generating cellular organelles designed to survive and contain their own unique genetic coding material, a circular mtDNA fragment of approximately 16,000 base pairs. The mitochondrial genetic system incorporates closely interacting bi-genomic factors encoded by the nuclear and mitochondrial genomes. Understanding the dynamics of mitochondrial genetics supporting mitochondrial biogenesis is especially important for the development of strategies for the treatment of rare and difficult-to-diagnose diseases. Gene therapy is one of the methods for correcting mitochondrial disorders.
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Enfermedades Mitocondriales , Humanos , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/terapia , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Terapia Genética , Patrón de HerenciaRESUMEN
Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94-58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.
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Dependovirus , Vectores Genéticos , Animales , Bovinos , Dependovirus/genética , Edición Génica/veterinaria , Lectinas Tipo C/genéticaRESUMEN
The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.
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Nucleótidos , Biosíntesis de Proteínas , Animales , Codón de Terminación/genética , Codón de Terminación/metabolismo , Eucariontes/metabolismo , Humanos , Mamíferos/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Extensión de la Cadena Peptídica de Translación , Terminación de la Cadena Péptídica Traduccional/genética , Factores de Terminación de Péptidos/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Adeno-associated virus (AAV) vectors have become an attractive tool for efficient gene transfer into animal tissues. Extensively studied as the vehicles for therapeutic constructs in gene therapy, AAVs are also applied for creating animal models of human genetic disorders. Neurological disorders are challenging to model in laboratory animals by transgenesis or genome editing, at least partially due to the embryonic lethality and the timing of the disease onset. Therefore, gene transfer with AAV vectors provides a more flexible option for simulating genetic neurological disorders. Indeed, the design of the AAV expression construct allows the reproduction of various disease-causing mutations, and also drives neuron-specific expression. The natural and newly created AAV serotypes combined with various delivery routes enable differentially targeting neuronal cell types and brain areas in vivo. Moreover, the same viral vector can be used to reproduce the main features of the disorder in mice, rats, and large laboratory animals such as non-human primates. The current review demonstrates the general principles for the development and use of AAVs in modeling neurological diseases. The latest achievements in AAV-mediated modeling of the common (e.g., Alzheimer's disease, Parkinson's disease, ataxias, etc.) and ultra-rare disorders affecting the central nervous system are described. The use of AAVs to create multiple animal models of neurological disorders opens opportunities for studying their mechanisms, understanding the main pathological features, and testing therapeutic approaches.
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High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (µDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Recent reports highlighted microutrophin (µUtrn) as a less immunogenic functional dystrophin substitute for gene therapy. In the current study, we created a human codon-optimized µUtrn which was subjected to side-by-side characterization with previously reported mouse and human µUtrn sequences after rAAV9 intramuscular injections in mdx mice. Long-term studies with systemic delivery of rAAV9-µUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to µDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized µUtrn overexpression. Furthermore, we verified that muscle-specific promoters MHCK7 and SPc5-12 drive a sufficient level of rAAV9-µUtrn expression to ameliorate the dystrophic phenotype in mdx mice. Our results provide ground for taking human codon-optimized µUtrn combined with muscle-specific promoters into clinical development as safe and efficient gene therapy for DMD.
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Codón , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Utrofina/uso terapéutico , Animales , Creatina Quinasa , Expresión Génica , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos mdx , Músculos/metabolismo , Fenotipo , Utrofina/administración & dosificación , Utrofina/genética , Utrofina/metabolismoRESUMEN
Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.
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Biosíntesis de Proteínas , SARS-CoV-2 , Proteínas no Estructurales Virales/fisiología , Animales , Sistema Libre de Células , Codón de Terminación/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Mutación , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Péptidos/química , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN Mensajero/metabolismo , Conejos , Ribosomas/metabolismoRESUMEN
Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using in vitro translation systems, we revealed that PDCD4 directly activates translation termination. PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions-binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.