65 research outputs found
Control of V(D)J Recombination through Transcriptional Elongation and Changes in Locus Chromatin Structure and Nuclear Organization
V(D)J recombination is the assembly of gene segments at the antigen receptor loci to
generate antigen receptor diversity in T and B lymphocytes. This process is regulated,
according to defined developmental programs, by the action of a single specific
recombinase complex formed by the recombination antigen gene (RAG-1/2) proteins
that are expressed in immature lymphocytes. V(D)J recombination is strictly controlled
by RAG-1/2 accessibility to specific recombination signal sequences in chromatin at
several levels: cellular lineage, temporal regulation, gene segment order, and allelic
exclusion. DNA cleavage by RAG-1/2 is regulated by the chromatin structure,
transcriptional elongation, and three-dimensional architecture and position of the
antigen receptor loci in the nucleus. Cis-elements specifically direct transcription and
V(D)J recombination at these loci through interactions with transacting factors that form
molecular machines that mediate a sequence of structural events. These events open
chromatin to activate transcriptional elongation and to permit the access of RAG-1/2 to
their recombination signal sequences to drive the juxtaposition of the V, D, and J
segments and the recombination reaction itself. This chapter summarizes the advances
in this area and the important role of the structure and position of antigen receptor loci
within the nucleus to control this process
Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease
Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.Fil: Suñé Pou, Marc. Universidad de Barcelona; EspañaFil: Limeres, MarĂa JosĂ©. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: Moreno Castro, Cristina. Institute of Parasitology and Biomedicine âLĂłpez-Neyra"; EspañaFil: HernĂĄndez Munain, Cristina. Institute of Parasitology and Biomedicine âLĂłpez-Neyra"; EspañaFil: Suñé Negre, Josep M.. Universidad de Barcelona; EspañaFil: Cuestas, MarĂa LujĂĄn. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: Suñé, Carlos. Institute of Parasitology and Biomedicine âLĂłpez-Neyra"; Españ
Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease
Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics
Targeting Splicing in the Treatment of Human Disease
The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event. Keywords: alternative splicing, precursor messenger RNA, therapy, genetic diseas
Comparative Analysis of the Physicochemical and Biological Characteristics of Freeze-Dried PEGylated Cationic Solid Lipid Nanoparticles
Cationic solid-lipid nanoparticles (cSLNs) have become a promising tool for gene and RNA therapies. PEGylation (PEG) is crucial in enhancing particle stability and protection. We evaluated the impact of PEG on the physicochemical and biological characteristics of cholesteryl-oleate cSLNs (CO-cSLNs). Several parameters were analyzed, including the particle size, polydispersity index, zeta potential, shape, stability, cytotoxicity, and loading efficiency. Five different formulations with specific PEGs were developed and compared in both suspended and freeze-dried states. Small, homogeneous, and cationic suspended nanoparticles were obtained, with the Gelucire 50/13 (PEG-32 hydrogenated palm glycerides; Gelucire) and DSPE-mPEG2000 (1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjungate-2000; DSPE) formulations exhibiting the smallest particle size (similar to 170 nm). Monodisperse populations of freeze-dried nanoparticles were also achieved, with particle sizes ranging from 200 to 300 nm and Z potential values of 30-35 mV. Notably, Gelucire again produced the smallest particle size (211.1 +/- 22.4), while the DSPE and Myrj S100 (polyoxyethylene (100) stearate; PEG-100 Stearate) formulations had similar particle sizes to CO-cSLNs (similar to 235 nm). The obtained PEGylated nanoparticles showed suitable properties: they were nontoxic, had acceptable morphology, were capable of forming SLNplexes, and were stable in both suspended and lyophilized states. These PEG-cSLNs are a potential resource for in vivo assays and have the advantage of employing cost-effective PEGs. Optimizing the lyophilization process and standardizing parameters are also recommended to maintain nanoparticle integrity
Dialysis is a key factor modulating interactions between critical process parameters during the microfluidic preparation of lipid nanoparticles
Manufacturing lipid nanoparticles through microfluidic mixing can be approached from a Quality by Design perspective. Research involving critical process parameters seems to focus on the total flow and flow rate ratio, thus other process variables, such as dialysis, are underestimated. This study used a Design of Experiments to identify the influence of critical process parameters on particle size, polydispersity index, and zeta potential. A response surface Design of Experiments modeled the influence of: total flow (400 to 4000 mu L min-1); flow rate ratio (3 to 9) and dialysis (yes/no). Results suggest that dialysis is a crucial parameter that strongly influences particle size and zeta potential and moderately affects polydispersity index. The flow rate ratio's relevance decreases when dialysis is performed. As the purification method can change the influence of other process parameters, it should be an integrated part of the microfluidic manufacturing of lipid nanoparticles instead of an extra step
Topical administration of bosentan prevents retinal neurodegeneration in experimental diabetes
Experimental evidence suggests that endothelin 1 (ET-1) is involved in the development of retinal microvascular abnormalities induced by diabetes. The effects of ET-1 are mediated by endothelin A- and B-receptors (ETA and ETB). Endothelin B-receptors activation mediates retinal neurodegeneration but there are no data regarding the effectiveness of ETB receptor blockage in arresting retinal neurodegeneration induced by diabetes. The main aim of the present study was to assess the usefulness of topical administration of bosentan (a dual endothelin receptor antagonist) in preventing retinal neurodegeneration in diabetic (db/db) mice. For this purpose, db/db mice aged 10 weeks were treated with one drop of bosentan (5 mg/mL, n = 6) or vehicle (n = 6) administered twice daily for 14 days. Six non-diabetic (db/+) mice matched by age were included as the control group. Glial activation was evaluated by immunofluorescence using specific antibodies against glial fibrillary acidic protein (GFAP). Apoptosis was assessed by TUNEL method. A pharmacokinetic study was performed in rabbits. We found that topical administration of bosentan resulted in a significant decrease of reactive gliosis and apoptosis. The results of the pharmacokinetic study suggested that bosentan reached the retina through the trans-scleral route. We conclude that topical administration of bosentan was effective in preventing neurodegeneration in the diabetic retina and, therefore, could be a good candidate to be tested in clinical trials
Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system
Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1â±â14.8ânm to 347.0â±â18.5ânm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.Centro de InvestigaciĂłn y Desarrollo en Fermentaciones Industriale
Evaluation of a Mixing versus a Cycling Strategy of Antibiotic Use in Critically-Ill Medical Patients: Impact on Acquisition of Resistant Microorganisms and Clinical Outcomes
OBJECTIVE: To compare the effect of two strategies of antibiotic
use (mixing vs. cycling) on the acquisition of resistant
microorganisms, infections and other clinical outcomes. METHODS:
Prospective cohort study in an 8-bed intensive care unit during
35- months in which a mixing-cycling policy of antipseudomonal
beta-lactams (meropenem, ceftazidime/piperacillin-tazobactam)
and fluoroquinolones was operative. Nasopharyngeal and rectal
swabs and respiratory secretions were obtained within 48h of
admission and thrice weekly thereafter. Target microorganisms
included methicillin-resistant S. aureus, vancomycin-resistant
enterococci, third-generation cephalosporin-resistant
Enterobacteriaceae and non-fermenters. RESULTS: A total of 409
(42%) patients were included in mixing and 560 (58%) in cycling.
Exposure to ceftazidime/piperacillin-tazobactam and
fluoroquinolones was significantly higher in mixing while
exposure to meropenem was higher in cycling, although overall
use of antipseudomonals was not significantly different
(37.5/100 patient-days vs. 38.1/100 patient-days). There was a
barely higher acquisition rate of microorganisms during mixing,
but this difference lost its significance when the cases due to
an exogenous Burkholderia cepacia outbreak were excluded (19.3%
vs. 15.4%, OR 0.8, CI 0.5-1.1). Acquisition of Pseudomonas
aeruginosa resistant to the intervention antibiotics or with
multiple-drug resistance was similar. There were no significant
differences between mixing and cycling in the proportion of
patients acquiring any infection (16.6% vs. 14.5%, OR 0.9, CI
0.6-1.2), any infection due to target microorganisms (5.9% vs.
5.2%, OR 0.9, CI 0.5-1.5), length of stay (median 5 d for both
groups) or mortality (13.9 vs. 14.3%, OR 1.03, CI 0.7-1.3).
CONCLUSIONS: A cycling strategy of antibiotic use with a 6-week
cycle duration is similar to mixing in terms of acquisition of
resistant microorganisms, infections, length of stay and
mortality
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