RESUMEN
Logic-gated engineered cells are an emerging therapeutic modality that can take advantage of molecular profiles to focus medical interventions on specific tissues in the body. However, the increased complexity of these engineered systems may pose a challenge for prediction and optimization of their behavior. Here we describe the design and testing of a flow cytometry-based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. In proof-of-concept experiments, this approach identifies inhibitory receptors that can operate as NOT gates when paired with activating receptors. The method may be used to generate large datasets to train machine learning models to better predict and optimize the function of logic-gated cell therapeutics.
RESUMEN
Neurons of the ventral tegmental area (VTA) are critical in the rewarding and reinforcing properties of drugs of abuse. Desensitization of VTA neurons to moderate extracellular concentrations of dopamine (DA) is dependent on protein kinase C (PKC) and intracellular calcium levels. This desensitization is called DA inhibition reversal, as it requires concurrent activation of D2 and D1-like receptors; activation of D2 receptors alone does not result in desensitization. Activation of other G-protein-linked receptors can substitute for D1 activation. Like D2 receptors, GABA(B) receptors in the VTA are coupled to G-protein-linked potassium channels. In the present study, we examined interactions between a GABA(B) agonist, baclofen, and dopamine agonists, dopamine and quinpirole, to determine whether there was some interaction in the processes of desensitization of GABA(B) and D2 responses. Long-duration administration of baclofen alone produced reversal of the baclofen-induced inhibition indicative of desensitization, and this desensitization persisted for at least 60 min after baclofen washout. Desensitization to baclofen was dependent on PKC. Dopamine inhibition was reduced for 30 min after baclofen-induced desensitization and conversely, the magnitude of baclofen inhibition was reduced for 30 min by long-duration application of dopamine, but not quinpirole. These results indicate that D2 and GABA(B) receptors share some PKC-dependent mechanisms of receptor desensitization.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Receptores de Dopamina D2/efectos de los fármacos , Receptores de GABA-B/efectos de los fármacos , Área Tegmental Ventral/efectos de los fármacos , Animales , Baclofeno/antagonistas & inhibidores , Baclofeno/farmacología , Carbazoles/farmacología , Recolección de Datos , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/fisiología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Técnicas In Vitro , Bombas de Infusión Implantables , Compuestos Organofosforados/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Quinpirol/farmacología , Ratas , Ratas Endogámicas F344 , Área Tegmental Ventral/citologíaRESUMEN
Dopaminergic neurons in the ventral tegmental area (VTA) are important for the rewarding properties of drugs of abuse, including ethanol. We previously demonstrated that ethanol excites VTA neurons and that ethanol reduces the amplitude of the after hyperpolarization (AHP) that follows spontaneous action potentials. Because the small conductance calcium-activated potassium current (SK) is a component of the AHP of VTA neurons, we assessed the effect of the SK blockers apamin and d-tubocurarine (d-TC) on the action of ethanol on dopaminergic VTA neurons with intracellular and extracellular recording in rat brain slices. Apamin (1-200 nM) and d-TC (100 and 400 microM) caused concentration-dependent reductions in the AHP amplitude. Ethanol (80 mM) caused a small reduction in the AHP. In the presence of apamin (40 nM), ethanol (80 mM) caused a much larger reduction in AHP amplitude. Extracellular studies showed that apamin (20, 40, and 100 nM) and d-TC (400 microM) had no significant effect on the spontaneous firing rate of dopaminergic VTA neurons but enhanced the potency of ethanol to increase their firing rate. These results indicate that the ethanol-induced reduction of the AHP and excitation of VTA neurons is not due to a reduction in SK current. Furthermore, blockade of SK current by apamin or d-TC enhances the excitatory effect of ethanol on dopaminergic VTA neurons. These data suggest that the amount of SK current present affects the sensitivity of dopaminergic VTA neurons to ethanol excitation and that neurotransmitters that reduce SK current may increase the reward potency of ethanol.
Asunto(s)
Dopamina/fisiología , Etanol/farmacología , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Canales de Potasio Calcio-Activados , Canales de Potasio , Área Tegmental Ventral/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Apamina/farmacología , Sinergismo Farmacológico , Técnicas In Vitro , Masculino , Neuronas/fisiología , Ratas , Ratas Endogámicas F344 , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Tubocurarina/farmacología , Área Tegmental Ventral/citología , Área Tegmental Ventral/fisiologíaRESUMEN
It is known that osteoblast precursor cells are found in the low-density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34- cells were cultured under conditions that promote growth and differentiation of mineral-secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34- cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal-specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell-derived osteoblasts displayed osteoblast-specific features. Functionally, these CD34+ cell-derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.
Asunto(s)
Antígenos CD34/análisis , Antígenos CD , Células de la Médula Ósea/inmunología , Osteoblastos/citología , Osteoblastos/inmunología , Células Madre/citología , Células Madre/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/inmunología , Antígenos de Diferenciación/análisis , Células de la Médula Ósea/citología , Recuento de Células , Diferenciación Celular/fisiología , Células Cultivadas , AMP Cíclico/biosíntesis , Dexametasona/farmacología , Humanos , Glicoproteínas de Membrana , Minerales/metabolismo , NAD+ Nucleosidasa/análisis , Osteoblastos/ultraestructura , Osteonectina/biosíntesis , Osteonectina/inmunología , Osteonectina/metabolismo , Hormona Paratiroidea/farmacología , Células Madre/ultraestructuraRESUMEN
The effect of long-term (1 y) low to excess ascorbic acid (AA) intake on bone mass was evaluated using guinea pigs that were 12-14 d old at the start of the experiment. Dietary AA was low (0.15 g/ kg diet) (n = 7), normal (0.50 g/kg) (n = 8) or excess (10 g/kg) (n = 8). After 12 mo, total body bone mineral density (BMD, mg/cm2) and bone mineral content (BMC, g) were determined by dual energy X-ray absorptiometry. Histomorphometric analysis of the cancellous bone of the proximal tibial metaphysis was completed after in vivo dual fluorochrome labeling. Total body BMD of the low AA group was 4.9% lower (P < 0.05), and total body BMC was 12.4% lower (P < 0.05) than in the normal AA group. Total body BMD and BMC were similar in normal and excess AA groups and in the low and excess AA groups. Histomorphometric analysis indicated significantly greater (P < 0.05) double-labeled bone surface, mineralizing surface, and bone formation rate in the low AA guinea pigs compared with the normal AA animals. Thus, there was greater bone turnover in the low AA group than in the normal AA guinea pigs. No differences in histomorphometric endpoints existed between the normal AA and excess AA groups. Long-term AA deficiency, during the period of rapid growth and slower phases of skeletal maturation, resulted in bone abnormalities in adult guinea pig skeletons. Long-term dietary AA excess caused no such abnormalities.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Densidad Ósea/efectos de los fármacos , Absorciometría de Fotón , Glándulas Suprarrenales/química , Animales , Ácido Ascórbico/análisis , Ácido Ascórbico/farmacología , Peso Corporal/fisiología , Calcio/sangre , Dieta , Relación Dosis-Respuesta a Droga , Femenino , Cobayas , Hígado/química , Tibia/efectos de los fármacos , Tibia/fisiología , Factores de TiempoRESUMEN
The effect of severe ascorbic acid deficiency on bone remodeling and collagen synthesis was evaluated in a 21 day experiment, using the scorbutic guinea pig model. Animals (n = 6-7/group) were assigned to one of three groups: scorbutic, pair-fed ascorbic acid-replete, or ad libitum ascorbic acid-replete groups. After 2 weeks, scorbutic animals started voluntarily decreasing food intake and losing weight. By day 19-21, at which time bone and tissue samples were collected and analyzed, scorbutic animals decreased food intake to 46% of usual and lost 9% body weight. Serum 25OHD3, 1,25(OH)2D3, calcium, and albumin were significantly lower (p < 0.05) in the scorbutic animals than in the other groups. Bone mineral density and bone mineral content of the proximal and central femur were significantly lower in the scorbutic group than in the other groups (p < 0.05). Morphometric analysis of tibia indicated significantly lower bone volume, fewer and thinner trabeculae, and a thinner growth plate in the scorbutic group, compared to the pair-fed and ad libitum groups (p < 0.05). Osteoclast surface was about 60% higher in the scorbutic group than in the pair-fed and ad libitum control groups (0.05 < p < 0.10). Mechanical strength of the femur and lumbar vertebral body tended to be lower when bone mass was altered in the same group. Collagen synthesis of articular cartilage and tendons was lower in the scorbutic group than in the pair-fed or ad libitum groups (p < 0.05). In conclusion, scurvy but not food restriction, per se, results in alterations in bone mass and tissue collagen synthesis.
Asunto(s)
Densidad Ósea/fisiología , Colágeno/biosíntesis , Escorbuto/metabolismo , Análisis de Varianza , Animales , Deficiencia de Ácido Ascórbico/fisiopatología , Fenómenos Biomecánicos , Peso Corporal/fisiología , Calcitriol/sangre , Calcio/sangre , Modelos Animales de Enfermedad , Ingestión de Alimentos/fisiología , Femenino , Fémur/metabolismo , Fémur/fisiología , Placa de Crecimiento/fisiología , Cobayas , Vértebras Lumbares/metabolismo , Vértebras Lumbares/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Escorbuto/fisiopatología , Albúmina Sérica/metabolismoRESUMEN
This study compares the attitudes of nonsecluded patients hospitalized on psychiatric units with either isolated or visible seclusion rooms. It was found that there were minimal differences between units. Patients at the hospital with visible seclusion rooms more often indicate that patients are often cured in such rooms, in contrast to the patients on the other unit who endorsed more stereotypical perceptions of the quiet room.