RESUMEN
Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.
Asunto(s)
ADN , Nanoestructuras , ADN/química , Nanoestructuras/química , Interacciones Hidrofóbicas e Hidrofílicas , Células Artificiales/química , Condensados Biomoleculares/químicaRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , G-Cuádruplex , Humanos , Demencia Frontotemporal/genética , Esclerosis Amiotrófica Lateral/genética , ARN/genética , ARN/química , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genéticaRESUMEN
Liver abscess was diagnosed in a man presenting with fever, chills and severe myopathy. The K. pneumoniae isolated from blood cultures belonged to the K1 serotype. The patient responded favourably to percutaneous drainage of the abscess and antibiotics. This is the first documented report of Klebsiella pneumoniae liver abscess syndrome (KLAS) described in Romania and may indicate the emergence of this syndrome in Eastern Europe.
Asunto(s)
Antígenos Bacterianos/análisis , Infecciones por Klebsiella/diagnóstico , Klebsiella pneumoniae/aislamiento & purificación , Absceso Hepático/complicaciones , Absceso Hepático/diagnóstico , Enfermedades Musculares/complicaciones , Enfermedades Musculares/diagnóstico , Anciano , Antibacterianos/uso terapéutico , Sangre/microbiología , Drenaje , Humanos , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/patología , Klebsiella pneumoniae/clasificación , Absceso Hepático/microbiología , Absceso Hepático/patología , Masculino , Enfermedades Musculares/microbiología , Enfermedades Musculares/patología , Polisacáridos Bacterianos , Radiografía Abdominal , Rumanía , Serogrupo , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl-xL can inhibit excitotoxicity in this model system. To track Bcl-xL expression, the gene for green fluorescent protein (GFP) was inserted in-frame, upstream of the Bcl-xL gene. The GFP-Bcl-xL gene was then cloned into an adeno-associated viral (AAV2) vector. GFP expression in both SH-SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl-xL expression in SH-SY5Y cells significantly reduced terminal deoxy-UTP nick-end labeling (TUNEL)-positive cells and maintained cell density after glutamate exposure. Similarly, Bcl-xL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV-mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy.
Asunto(s)
Dependovirus/fisiología , Técnicas de Transferencia de Gen , Enfermedad de la Neurona Motora/prevención & control , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Western Blotting/métodos , Caspasa 3 , Caspasas/metabolismo , Recuento de Células/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Expresión Génica/fisiología , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/fisiología , Ácido Glutámico/efectos adversos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica/métodos , Etiquetado Corte-Fin in Situ/métodos , Enfermedad de la Neurona Motora/genética , Neuroblastoma , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Transfección/métodosRESUMEN
The present study examines gene delivery to cultured motor neurons (MNs) with the Rabies G protein (RabG)-pseudotyped lentiviral equine infectious anemia virus (RabG.EIAV) vector. RabG.EIAV-mediated beta-galactosidase (RabG.EIAV-LacZ) gene expression in cultured MNs plateaus 120 h after infection. The rate and percent of gene expression observed are titer-dependent (P < 0.001). The rat IGF-I cDNA sequence was then cloned into a RabG.EIAV vector (RabG.EIAV-IGF-I) and was shown to induce IGF-I expression in HEK 293 cells. MNs infected with RabG.EIAV-IGF-I demonstrate enhanced survival compared to MNs infected with RabG.EIAV-LacZ virus (P < 0.01). In addition, IGF-I expression in cultured MNs induced profound MN axonal elongation compared to control virus (P < 0.01). The enhanced motor neuron tropism of RabG.EIAV previously demonstrated in vivo, together with the trophic effects of RabG.EIAV-IGF-I MN gene expression may lend this vector to therapeutic application in motor neuron disease.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Virus de la Anemia Infecciosa Equina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Neuronas Motoras/metabolismo , Factores de Crecimiento Nervioso/genética , Animales , Supervivencia Celular/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Terapia Genética/métodos , Conos de Crecimiento/metabolismo , Conos de Crecimiento/virología , Humanos , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/terapia , Fragmentos de Péptidos/genética , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Receptor IGF Tipo 1/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismo , Médula Espinal/virología , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genéticaRESUMEN
A novel peptide with the binding characteristics of tetanus toxin was identified with phage display, for application in therapeutic protein and vector motor and sensory neuron targeting. A 12mer phage library was biopanned on trisialoganglioside (G(T1b)) and eluted with the tetanus toxin C fragment (rTTC). Phage ELISAs revealed increases in G(T1b) binding for the Tet1 and Tet2 phage clones when compared to peptideless phage (PLP). rTTC displaced both Tet1 and Tet2 phage clones from G(T1b), and both clones reduced rTTC-G(T1b) binding. Comparison of Tet1, Tet2, PLP, and the random phage library binding to PC12 and HEK293 cells revealed enhanced cellular binding by Tet1 and Tet2 phage. Tet1 phage binding was selective for neurons. Immunofluorescence also confirmed selective PC12 binding of Tet1 and Tet2 phage. Fluorescein-conjugated synthetic Tet1, but not Tet2, peptide showed strong binding to cultured PC12, primary motor neurons, and dorsal root ganglion (DRG) cells. Synthetic Tet1 bound DRG and motor neurons but not muscle in tissue sections. The enhanced neuronal binding affinity and specificity of Tet1, a novel 12 amino acid peptide, suggests potential utility for targeting neurotherapeutic proteins and viral vectors in the treatment of motor neuron disease, neuropathy, and pain.
Asunto(s)
Gangliósidos/metabolismo , Neuronas Motoras/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Toxina Tetánica/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Encéfalo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/metabolismo , Humanos , Imitación Molecular/fisiología , Datos de Secuencia Molecular , Células PC12 , Péptidos/genética , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Médula Espinal/metabolismo , Toxina Tetánica/químicaRESUMEN
BACKGROUND: Adenosine modulates cell excitability, acetylcholine release, nociception, and sleep. Pontine cholinergic neurotransmission contributes to the generation and maintenance of electroencephalographic and behavioral arousal. Adenosine A(1) receptors inhibit arousal-promoting, pontine cholinergic neurons, and adenosine enhances sleep. No previous studies have determined whether pontine adenosine also modulates recovery from anesthesia. Therefore, the current study tested the hypotheses that dialysis delivery of the adenosine A(1) receptor agonist N6-p-sulfophenyladenosine (SPA) into the pontine reticular formation would decrease acetylcholine release and increase the time needed for recovery from halothane anesthesia. METHODS: A microdialysis probe was positioned in the pontine reticular formation of halothane-anesthetized cats. Probes were perfused with Ringer's solution (control) followed by the adenosine A(1) receptor agonist SPA (0.088 or 8.8 mm). Dependent measures included acetylcholine release and a numeric assessment of recovery from anesthesia. An intensive, within-subjects design and analysis of variance evaluated SPA's main effect on acetylcholine release and anesthetic recovery. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 microm) was coadministered with SPA to test for antagonist blocking of SPA's effects. RESULTS: SPA significantly (P < 0.0001) decreased acetylcholine release in the pontine reticular formation and significantly (P < 0.0001) delayed recovery from anesthesia. Coadministration of SPA and DPCPX caused no decrease in acetylcholine release or delay in postanesthetic recovery. Dialysis delivery of SPA into the cerebellar cortex confirmed that the SPA effects were site-specific to the pontine reticular formation. CONCLUSION: The results provide a novel extension of the sleep-promoting effects of adenosine by showing that pontine delivery of an adenosine A(1) receptor agonist delays resumption of wakefulness following halothane anesthesia. This extension is consistent with a potentially larger relevance of the current findings for efforts to specify neurons and molecules causing physiologic and behavioral traits comprising anesthetic states. These data support the conclusion that adenosine A(1) receptors in medial regions of the pontine reticular formation, known to modulate sleep, also contribute to the generation and/or maintenance of halothane anesthesia.
Asunto(s)
Acetilcolina/metabolismo , Periodo de Recuperación de la Anestesia , Puente/efectos de los fármacos , Agonistas del Receptor Purinérgico P1 , Formación Reticular/efectos de los fármacos , Estimulación Acústica , Animales , Gatos , Cromatografía Líquida de Alta Presión , Electroquímica , Electroencefalografía/efectos de los fármacos , Electromiografía/efectos de los fármacos , Electrooculografía/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Microdiálisis , Actividad Motora/efectos de los fármacos , Oxígeno/sangre , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Xantinas/farmacologíaRESUMEN
BACKGROUND: Both pain and the pharmacologic management of pain can cause the undesirable effect of sleep disruption. One goal of basic and clinical neuroscience is to facilitate rational drug development by identifying the brain regions and neurochemical modulators of sleep and pain. Adenosine is thought to be an endogenous sleep promoting substance and adenosinergic compounds can contribute to pain management. In the pontine brain stem adenosine promotes sleep but the effects of pontine adenosine on pain have not been studied. This study tested the hypothesis that an adenosine agonist would cause antinociception when microinjected into pontine reticular formation regions that regulate sleep. METHODS: The tail flick latency (TFL) test quantified the time in seconds for an animal to move its tail away from a thermal stimulus created by a beam of light. TFL measures were used to evaluate the antinociceptive effects of the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA). Pontine microinjection of SPA (0.1 microg/0.25 microl, 0.88 mm) was followed by TFL measures as a function of time after drug delivery and across the sleep-wake cycle. RESULTS: Compared with saline (control), pontine administration of the adenosine agonist significantly increased latency to tail withdrawal (P < 0.0001). The increase in antinociceptive behavior evoked by the adenosine agonist SPA was blocked by pretreatment with the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.75 ng/0.25 microl, 10 microm). CONCLUSIONS: These preclinical data encourage additional research on the cellular mechanisms by which adenosine in the pontine reticular formation contributes to the supraspinal modulation of pain.