RESUMEN
The orbitofrontal cortex (OFC) is a brain region involved in higher-order decision-making. Rodent studies show that cocaine self-administration (CSA) reduces OFC contribution to goal-directed behavior and behavioral strategies to avoid drug intake. This change in OFC function persists for many weeks after cocaine withdrawal, suggesting involvement in the process of addiction. The mechanisms underlying impaired OFC function by cocaine are not well-understood. However, studies implicate altered OFC serotonin (5-HT) function in disrupted cognitive processes during addiction and other psychiatric disorders. Thus, it is hypothesized that cocaine impairment of OFC function involves changes in 5-HT signaling, and previous work shows that 5-HT1A and 5-HT2A receptor-mediated effects on OFC pyramidal neurons (PyNs) are impaired weeks after cocaine withdrawal. However, 5-HT effects on other contributors to OFC circuit function have not been fully investigated, including the parvalbumin-containing, fast-spiking interneurons (OFCPV), whose function is essential to normal OFC-mediated behavior. Here, 5-HT function in naive rats and those withdrawn from CSA were evaluated using a novel rat transgenic line in which the rat parvalbumin promoter drives Cre-recombinase expression to permit identification of OFCPV cells by fluorescent reporter protein expression. We find that whereas CSA altered basal synaptic and membrane properties of the OFCPV neurons in a sex-dependent manner, the effects of 5-HT on these cells were unchanged by CSA. These data suggest that the behavioral effects of dysregulated OFC 5-HT function caused by cocaine experience are primarily mediated by changes in 5-HT signaling at PyNs, and not at OFCPV neurons.
Asunto(s)
Cocaína , Animales , Integrasas , Neuronas , Parvalbúminas , Corteza Prefrontal , Ratas , SerotoninaRESUMEN
Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.
Asunto(s)
Células Madre Germinales Adultas/metabolismo , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Neuronas Dopaminérgicas/metabolismo , Edición Génica/métodos , Marcación de Gen/métodos , Animales , Proteína 9 Asociada a CRISPR/genética , Desoxirribonucleasa I/genética , Dependovirus , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes , Vectores Genéticos , Integrasas , Proteínas Luminiscentes/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida , Ratas , Ratas Transgénicas , Tirosina 3-Monooxigenasa/genética , Proteína Fluorescente RojaRESUMEN
Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiología , Edición Génica/métodos , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Células HEK293 , Duplicado del Terminal Largo de VIH/genética , Duplicado del Terminal Largo de VIH/fisiología , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Lipopolisacáridos/farmacología , Mutación/genética , Provirus/genética , Provirus/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismoRESUMEN
Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.
Asunto(s)
VIH-1/fisiología , Microglía/virología , Provirus/fisiología , Virión/fisiología , Antiinfecciosos/farmacología , Sistemas CRISPR-Cas , Línea Celular , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Lipopolisacáridos/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Microglía/efectos de los fármacos , Microglía/metabolismo , Provirus/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulfasalazina/farmacología , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/farmacología , Virión/genéticaRESUMEN
OBJECTIVE: To create a nomogram of fetal clavicle length (CL) throughout gestation. METHODS: Cross-sectional study of patients between 14 and 42 weeks' gestation. Inclusion criteria consisted of well-established dates (consistent with early ultrasound), singleton, non-anomalous fetuses, and intact amniotic membranes. Sonographic measurements included biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL), humerus length (HL) and sonographically estimated fetal weight (SEFW). For every case, the average of three separate measurements of the CL was used. The 5th, 50th and 95th centiles were obtained by least squares regression. Pearson's correlation coefficient and associated P-values for the relationships between CL and other biometric measurements were calculated. The data were compared to a nomogram of the CL generated in 1985 from the measurement of 85 fetuses. RESULTS: A total of 623 consecutive patients were studied. In all but three cases, CL was successfully measured. Mean maternal age was 27.7 +/- 6.2 years, median gravidity 3 (range, 1-14) and median parity 1 (range, 0-9). Mean CL (mm) = -75.30 + 32.70*ln(GA) and SD = -0.41 + 0.08328*GA, where ln represents the natural logarithm and GA the gestational age in weeks. Fetal CL correlated significantly and strongly with BPD, HC, AC, HL, FL and the logarithm of SEFW, with Pearson correlation values of 0.973, 0.977, 0.976, 0.979, 0.977 and 0.979, respectively (all P < 0.001). Measurements according to comparable 1985 data were consistently substantially below the present data (smaller CL for any given GA except below 17 weeks' gestation). CONCLUSIONS: We propose a new nomogram of CL, which differs significantly from the previously published nomogram. We suggest that the present data reflect the use of high-resolution ultrasound technology and propose that these data, based on a large number of fetuses, replace the previous nomogram. We also suggest that the '1 mm = 1 week' rule of thumb should no longer be used, since it can be erroneous by as much as 6 weeks.
Asunto(s)
Clavícula/embriología , Nomogramas , Adulto , Clavícula/diagnóstico por imagen , Estudios Transversales , Femenino , Desarrollo Fetal , Edad Gestacional , Humanos , Embarazo , Ultrasonografía PrenatalRESUMEN
PepR1 from Lactobacillus delbrueckii subsp bulgaricus (Lb. bulgaricus) is involved in biosynthesis regulation of the prolidase PepQ. In this paper, we demonstrated that Lb. bulgaricus PepR1 biosynthesis is not constitutive like those of several bacteria but is auto-regulated and depends on the glucose concentration of the culture medium. We propose a model for PepQ regulation by PepR1.
Asunto(s)
Proteínas Bacterianas , Lactobacillus/metabolismo , Transactivadores/biosíntesis , Proteínas de Unión al ADN , Dipeptidasas/metabolismo , Homeostasis , Regiones Promotoras Genéticas , Proteínas Represoras , Elementos de Respuesta , Transactivadores/genéticaRESUMEN
UNLABELLED: The subdural hematoma detected in utero is unusual but probably under-diagnosed and has a bad prognostic. CASE REPORT: A newborn had a subdural hematoma detected with a prenatal ultrasonography at 31 weeks' gestation, probably in keeping with the regular treatment of AAS. Diagnosis was possible through antenatal MRI at 34 weeks' gestation. The mother chose to follow her pregnancy. The operation at day 3 had no complications and neurological progress at 12 months was very good. CONCLUSION: A subdural hematoma detected in utero may have a good prognostic. Prenatal MRI is useful for the check-up. However, medical and ethical problems remain in these cases.