RESUMEN

OBJECTIVE: This study evaluated the effects of cessation of both conventional low-frequency (50 Hz) and high-frequency (10 kHz) spinal cord stimulation (SCS) on the cardiospinal neural network activity in pigs with myocardial infarction (MI). The objective is to provide an insight into the memory effect of SCS. MATERIALS AND METHODS: In nine Yorkshire pigs, chronic MI was created by delivering microspheres to the left circumflex coronary artery. Five weeks after MI, anesthetized pigs underwent sternotomy to expose the heart for performing acute ischemia intervention, and laminectomy to expose the T1-T4 spinal regions for extracellular in vivo neural recording and SCS. Cardiac ischemic-sensitive neurons were identified by selective responsiveness to left anterior descending (LAD) coronary artery occlusion. SCS episodes were delivered in a random order between low- (50 Hz) and high- (10 kHz) frequency, for 1 minute, at 90% of the motor threshold current. Neural firing and synchrony of ischemic-sensitive spinal neurons were evaluated before vs after SCS. RESULTS: Using a 64-channel microelectrode array, 2711 spinal neurons were recorded extracellularly. LAD ischemia excited 228 neurons that were labeled as ischemic-responsive neurons. The cessation of 50-Hz SCS caused a higher activation than did inhibition of ischemic-responsive neurons (41 activated vs 19 inhibited), whereas the cessation of 10-kHz SCS caused an opposite response with higher inhibition (11 activated vs 28 inhibited, p < 0.01 vs 50 Hz). Termination of low-frequency SCS caused an increase in ischemic-responsive neuronal firing rate compared with high-frequency SCS (50 Hz: 0.39 Hz ± 0.16 Hz, 10 kHz: -0.11 Hz ± 0.057 Hz, p < 0.01). In addition, SCS delivered at 50 Hz increased the number of synchronized pairs of neurons by 205 pairs, whereas high-frequency SCS decreased the number of synchronized pairs by 345 pairs (p < 0.01). CONCLUSIONS: High-frequency (10 kHz) stimulation provides persistent suppression of the ischemia-sensitive neurons after termination of SCS. In contrast, the spinal neural network reverted to excitatory state after termination of low-frequency (50 Hz) stimulation.

RESUMEN

Introduction: Myocardial ischemia disrupts the cardio-spinal neural network that controls the cardiac sympathetic preganglionic neurons, leading to sympathoexcitation and ventricular tachyarrhythmias (VTs). Spinal cord stimulation (SCS) is capable of suppressing the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not fully known. Methods: In this pre-clinical study, we investigated the impact of SCS on the spinal neural network in mitigating myocardial ischemia-induced sympathoexcitation and arrhythmogenicity. Ten Yorkshire pigs with left circumflex coronary artery (LCX) occlusion-induced chronic myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy at 4-5 weeks post-MI. The activation recovery interval (ARI) and dispersion of repolarization (DOR) were analyzed to evaluate the extent of sympathoexcitation and arrhythmogenicity during the left anterior descending coronary artery (LAD) ischemia. Extracellular in vivo and in situ spinal dorsal horn (DH) and intermediolateral column (IML) neural recordings were performed using a multichannel microelectrode array inserted at the T2-T3 segment of the spinal cord. SCS was performed for 30 min at 1 kHz, 0.03 ms, 90% motor threshold. LAD ischemia was induced pre- and 1 min post-SCS to investigate how SCS modulates spinal neural network processing of myocardial ischemia. DH and IML neural interactions, including neuronal synchrony as well as cardiac sympathoexcitation and arrhythmogenicity markers were evaluated during myocardial ischemia pre- vs. post-SCS. Results: ARI shortening in the ischemic region and global DOR augmentation due to LAD ischemia was mitigated by SCS. Neural firing response of ischemia-sensitive neurons during LAD ischemia and reperfusion was blunted by SCS. Further, SCS showed a similar effect in suppressing the firing response of IML and DH neurons during LAD ischemia. SCS exhibited a similar suppressive impact on the mechanical, nociceptive and multimodal ischemia sensitive neurons. The LAD ischemia and reperfusion-induced augmentation in neuronal synchrony between DH-DH and DH-IML pairs of neurons were mitigated by the SCS. Discussion: These results suggest that SCS is decreasing the sympathoexcitation and arrhythmogenicity by suppressing the interactions between the spinal DH and IML neurons and activity of IML preganglionic sympathetic neurons.

RESUMEN

RNA hybridization-based spatial transcriptomics provides unparalleled detection sensitivity. However, inaccuracies in segmentation of image volumes into cells cause misassignment of mRNAs which is a major source of errors. Here, we develop JSTA, a computational framework for joint cell segmentation and cell type annotation that utilizes prior knowledge of cell type-specific gene expression. Simulation results show that leveraging existing cell type taxonomy increases RNA assignment accuracy by more than 45%. Using JSTA, we were able to classify cells in the mouse hippocampus into 133 (sub)types revealing the spatial organization of CA1, CA3, and Sst neuron subtypes. Analysis of within cell subtype spatial differential gene expression of 80 candidate genes identified 63 with statistically significant spatial differential gene expression across 61 (sub)types. Overall, our work demonstrates that known cell type expression patterns can be leveraged to improve the accuracy of RNA hybridization-based spatial transcriptomics while providing highly granular cell (sub)type information. The large number of newly discovered spatial gene expression patterns substantiates the need for accurate spatial transcriptomic measurements that can provide information beyond cell (sub)type labels.

Asunto(s)

Perfilación de la Expresión Génica , Transcriptoma , Animales , Simulación por Computador , Ratones , Neuronas , ARN Mensajero , Transcriptoma/genética

RESUMEN

Balancing cell death is essential to maintain healthy tissue homeostasis and prevent disease. Tumor necrosis factor (TNF) not only activates nuclear factor κB (NFκB), which coordinates the cellular response to inflammation, but may also trigger necroptosis, a pro-inflammatory form of cell death. Whether TNF-induced NFκB affects the fate decision to undergo TNF-induced necroptosis is unclear. Live-cell microscopy and model-aided analysis of death kinetics identified a molecular circuit that interprets TNF-induced NFκB/RelA dynamics to control necroptosis decisions. Inducible expression of TNFAIP3/A20 forms an incoherent feedforward loop to interfere with the RIPK3-containing necrosome complex and protect a fraction of cells from transient, but not long-term TNF exposure. Furthermore, dysregulated NFκB dynamics often associated with disease diminish TNF-induced necroptosis. Our results suggest that TNF's dual roles in either coordinating cellular responses to inflammation, or further amplifying inflammation are determined by a dynamic NFκB-A20-RIPK3 circuit, that could be targeted to treat inflammation and cancer.

Asunto(s)

FN-kappa B/metabolismo , Necroptosis , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Inflamación/patología , Cinética , Ratones , Modelos Biológicos , Necroptosis/efectos de los fármacos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Gene expression variability, differences in the number of mRNA per cell across a population of cells, is ubiquitous across diverse organisms with broad impacts on cellular phenotypes. The role of chromatin in regulating average gene expression has been extensively studied. However, what aspects of the chromatin contribute to gene expression variability is still underexplored. Here we addressed this problem by leveraging chromatin diversity and using a systematic investigation of randomly integrated expression reporters to identify what aspects of chromatin microenvironment contribute to gene expression variability. Using DNA barcoding and split-pool decoding, we created a large library of isogenic reporter clones and identified reporter integration sites in a massive and parallel manner. By mapping our measurements of reporter expression at different genomic loci with multiple epigenetic profiles including the enrichment of transcription factors and the distance to different chromatin states, we identified new factors that impact the regulation of gene expression distributions.

Asunto(s)

Cromatina/genética , Regulación de la Expresión Génica/genética , Cromatina/metabolismo , Epigénesis Genética/genética , Epigenómica/métodos , Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genoma/genética , Genómica/métodos , Humanos , Células K562 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Electroacupuncture (EA) is widely used as an effective method to treat stress-related disorders. However, its mechanisms remain largely unknown. The aim of this study was to investigate the effects and mechanisms of EA on gastric slow wave (GSW) dysrhythmia and c-Fos expression in the nucleus of the solitary tract (NTS) induced by stress in a rodent model of functional dyspepsia (FD). Rats in the neonatal stage were treated using intragastric iodoacetamide. Eight weeks later, the rats were implanted with electrodes in the stomach for the measurement of GSW and electrodes into accupoints ST36 for EA. Autonomic functions were assessed by spectral analysis of heart rate variability. Rats were placed for 30 min in a cylindrical plastic tube for acute restraint stress. The involvement of a central afferent pathway was assessed by measuring c-Fos-immunoreactive cells in the NTS. 1) EA normalized restraint stress-induced impairment of GSW in FD rats. 2) EA significantly increased vagal activity (P = 0.002) and improved sympathovagal balance (P = 0.004) under stress in FD rats. 3) In FD rats under restraint stress, plasma norepinephrine concentration was increased substantially (P < 0.01), which was suppressed with EA. 4) The EA group showed increased c-Fos-positive cell counts in the NTS compared with the sham EA group (P < 0.05) in FD rats. Acute restraint stress induces gastric dysrhythmia in a rodent model of FD. EA at ST36 improves GSW under stress in FD rats mediated via the central and autonomic pathways, involving the NTS and vagal efferent pathway.

Asunto(s)

Sistema Nervioso Autónomo/fisiopatología , Sistema Nervioso Central/fisiopatología , Dispepsia/fisiopatología , Dispepsia/terapia , Electroacupuntura , Gastropatías/terapia , Estrés Psicológico/complicaciones , Vías Aferentes/fisiopatología , Animales , Animales Recién Nacidos , Vaciamiento Gástrico , Yodoacetamida , Masculino , Norepinefrina/sangre , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Ratas , Ratas Sprague-Dawley , Restricción Física , Núcleo Solitario/metabolismo , Gastropatías/inducido químicamente , Nervio Vago/fisiopatología

RESUMEN

Gene expression variability in mammalian systems plays an important role in physiological and pathophysiological conditions. This variability can come from differential regulation related to cell state (extrinsic) and allele-specific transcriptional bursting (intrinsic). Yet, the relative contribution of these two distinct sources is unknown. Here, we exploit the qualitative difference in the patterns of covariance between these two sources to quantify their relative contributions to expression variance in mammalian cells. Using multiplexed error robust RNA fluorescent in situ hybridization (MERFISH), we measured the multivariate gene expression distribution of 150 genes related to Ca2+ signaling coupled with the dynamic Ca2+ response of live cells to ATP. We show that after controlling for cellular phenotypic states such as size, cell cycle stage, and Ca2+ response to ATP, the remaining variability is effectively at the Poisson limit for most genes. These findings demonstrate that the majority of expression variability results from cell state differences and that the contribution of transcriptional bursting is relatively minimal.

Asunto(s)

Señalización del Calcio , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , Ciclo Celular , Línea Celular , Tamaño de la Célula , Femenino , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Análisis de la Célula Individual , Biología de Sistemas/métodos

RESUMEN

Impaired gastric accommodation (GA) has been frequently reported in various gastrointestinal diseases. No standard treatment strategy is available for treating impaired GA. We explored the possible effect of sacral nerve stimulation (SNS) on GA and discovered a spinal afferent and vagal efferent mechanism in rats. Sprague-Dawley rats (450-500 g) with a chronically implanted gastric cannula and ECG electrodes were studied in a series of sessions to study: 1) the effects of SNS with different parameters on gastric tone, compliance, and accommodation using a barostat device; two sets of parameters were tested as follows: parameter 1) 5 Hz, 500 µs, 10 s on 90 s off; 90% motor threshold and parameter 2) same as parameter 1 but 25 Hz; 2) the involvement of spinal afferent pathway via detecting c-fos immunoreactive (IR) cells in the nucleus of the solitary tract (NTS) of the brain; 3) the involvement of vagal efferent activity via the spectral analysis of heart rate variability derived from the ECG; and 4) the nitrergic mechanism, Nω-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase (NOS) inhibitor, was given before SNS at 5 Hz. Compared with sham-SNS: 1) SNS at 5 Hz inhibited gastric tone and increased gastric compliance and GA. No difference was noted between the stimulation frequencies of 5 and 25 Hz. 2) SNS increased the expression of c-fos in the NTS. 3) SNS increased cardiac vagal efferent activity and decreased the sympathovagal ratio. 4) l-NAME blocked the relaxation effect of SNS. In conclusion, SNS with certain parameters relaxes gastric fundus and improves gastric accommodation mediated via a spinal afferent and vagal efferent pathway.NEW & NOTEWORTHY Currently, there is no adequate medical therapy for impaired gastric accommodation, since medications that relax the fundus often impair antral peristalsis and thus further delay gastric emptying that is commonly seen in patients with functional dyspepsia or gastroparesis. The advantage of the potential sacral nerve stimulation therapy is that it improves gastric accommodation by enhancing vagal activity, and the enhanced vagal activity would lead to enhanced antral peristalsis rather than inhibiting it.

Asunto(s)

Terapia por Estimulación Eléctrica/métodos , Vaciamiento Gástrico , Plexo Lumbosacro/fisiología , Neuronas Nitrérgicas/fisiología , Reflejo , Nervios Espinales/fisiología , Estómago/inervación , Nervio Vago/fisiología , Vías Aferentes/fisiología , Animales , Vías Eferentes/fisiología , Gastroparesia/fisiopatología , Gastroparesia/terapia , Masculino , Ratas Sprague-Dawley

RESUMEN

The purpose of this study was to define the mechanism by which cardiac neuraxial decentralization or spinal cord stimulation (SCS) reduces ischemia-induced ventricular fibrillation (VF). Direct measurements of norepinephrine (NE) levels in the left ventricular interstitial fluid (ISF) by microdialysis, in response to transient (15-minute) coronary artery occlusion (CAO), were performed in anesthetized canines. Responses were studied in animals with intact neuraxes and were compared with those in which the intrathoracic component of the cardiac neuraxes (stellate ganglia) or the intrinsic cardiac neuronal (ICN) system was surgically delinked from the central nervous system and those with intact neuraxes with preemptive SCS (T1-T3). With intact neuraxes, animals with exaggerated NE release due to CAO were at increased risk for VF. During CAO, there was a 152% increase in NE when the neuraxes were intact compared with 114% following stellate decentralization and 16% following ICN decentralization. During SCS, CAO NE levels increased by 59%. Risk for CAO-induced VF was 38% in controls, 8% following decentralization, and 11% following SCS. These data indicate that ischemia-related afferent neuronal transmission differentially engages central and intrathoracic sympathetic reflexes and amplifies sympathoexcitation. Differences in regional ventricular NE release are associated with increased risk for VF. Surgical decentralization or SCS reduced NE release and VF.

Asunto(s)

Corazón/fisiología , Isquemia/metabolismo , Norepinefrina/metabolismo , Estimulación de la Médula Espinal/métodos , Simpatectomía/métodos , Fibrilación Ventricular/metabolismo , Animales , Arritmias Cardíacas , Modelos Animales de Enfermedad , Perros , Femenino , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/cirugía , Isquemia/cirugía , Masculino , Sistema Nervioso/patología , Reflejo , Ganglio Estrellado/metabolismo , Transmisión Sináptica , Fibrilación Ventricular/cirugía

RESUMEN

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Asunto(s)

Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Femenino , Células HeLa , Humanos , Masculino

RESUMEN

Although sacral nerve stimulation (SNS) has been applied for treating constipation, its parameters were adopted from SNS for fecal incontinence, its effects are limited, and mechanisms are largely unknown. We investigated the effects and mechanism of SNS with appropriate parameters on constipation in rats treated with loperamide. First, using rectal compliance as an outcome measure, an experiment was performed to derive effective SNS parameters. Then, a 7-day SNS was performed in rats with constipation induced by loperamide. Autonomic functions were assessed by spectral analysis of heart rate variability (HRV) derived from an electrocardiogram. Serum levels of pancreatic polypeptide (PP), norepinephrine (NE), and acetylcholine (ACh) in colon were assessed. 1) Acute SNS at 5 Hz, 100 µs was found effective in enhancing rectal compliance and accelerating distal colon transit (P < 0.05 vs. sham SNS). 2) The 7-day SNS normalized loperamide-induced constipation, assessed by the number, weight, and water content of fecal pellets, and accelerated the distal colon transit (29.4 ± 3.7 min with sham SNS vs. 16.4 ± 5.3 min with SNS but not gastric emptying or intestinal transit. 3) SNS significantly increased vagal activity (P = 0.035) and decreased sympathetic activity (P = 0.012), assessed by spectral analysis of HRV as well as by the serum PP. 4) SNS increased ACh in the colon tissue; atropine blocked the accelerative effect of SNS on distal colon transit. We concluded that SNS with appropriate parameters improves constipation induced by loperamide by accelerating distal colon motility, mediated via the autonomic-cholinergic function.NEW & NOTEWORTHY Although sacral nerve stimulation (SNS) has been applied for treating constipation, its parameters were adopted from SNS for fecal incontinence, effects are limited, and mechanisms are largely unknown. This paper shows that SNS with appropriate parameters improves constipation induced by loperamide by accelerating distal colon motility mediated via the autonomic-cholinergic function.

Asunto(s)

Colon/fisiología , Estreñimiento/terapia , Terapia por Estimulación Eléctrica/métodos , Tránsito Gastrointestinal , Plexo Lumbosacro/fisiología , Acetilcolina/metabolismo , Animales , Sistema Nervioso Autónomo/fisiología , Colon/inervación , Colon/metabolismo , Estreñimiento/etiología , Loperamida/toxicidad , Masculino , Norepinefrina/sangre , Polipéptido Pancreático/sangre , Ratas , Ratas Sprague-Dawley

RESUMEN

BACKGROUND: Acupuncture has been widely accepted for treatments of many diseases. This study was performed to determine effects and mechanisms of electroacupuncture (EA) by chronically implanted electrodes at acupoint ST36 on colonic inflammation induced by TNBS in rats. METHODS: After intrarectal administration of TNBS, the rats were treated with sham-EA, EA1/EA2 (two sets of parameters) for 3 weeks. Disease activity index (DAI), macroscopic and microscopic lesions, plasma levels of TNF-α, IL-1ß and IL-6 were observed as evaluation of inflammatory responses. The autonomic function was assessed by analysis of the heart rate variability. RESULTS: (a) Vagal activity was significantly increased with both acute and chronic EA1/EA2; (b) DAI was significantly decreased with both chronic EA1 and EA2, and EA2 was more potent than EA1 (P < 0.05); (c) The macroscopic score was 6.4 ± 0.6 with sham-EA and reduced to 4.9 ± 0.1 with EA1 (P < 0.05) and 4.0 ± 0.2 with EA2 (all P < 0.05). The histological score was 4.05 ± 0.58 with sham-EA and remained unchanged (3.71 ± 0.28) with EA1 (P > 0.05) but reduced to 3.0 ± 0.3 with EA2 (P < 0.01); (d) The plasma levels of TNF-α, IL-1ß and IL-6 were significantly decreased with EA2. CONCLUSIONS: Electrical stimulation at ST36 improves colonic inflammation in TNBS-treated rats by inhibiting pro-inflammatory cytokines via the autonomic mechanism.

Asunto(s)

Sistema Nervioso Autónomo/fisiopatología , Colitis/fisiopatología , Electroacupuntura , Puntos de Acupuntura , Animales , Citocinas , Modelos Animales de Enfermedad , Inflamación/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Nervio Vago/fisiopatología

RESUMEN

BACKGROUND/AIMS: Postoperative ileus increases healthcare costs and reduces the postoperative quality of life (QOL). The aim of this study is to investigate effects and mechanisms of electroacupuncture (EA) at ST36 and PC6 on gastrointestinal motility in rat model of postoperative ileus. METHODS: Laparotomy was performed in 24 rats (control [n = 8], sham-EA [n = 8], and EA [n = 8]) for the implantation of electrodes in the stomach and mid-jejunum for recording of gastric and small intestinal slow waves. Electrodes were placed in the chest skin for electrocardiogram (ECG). Intestinal manipulation (IM) was performed in Sham-EA and EA rats after surgical procedures. Small intestinal transit (SIT), gastric emptying (GE), postoperative pain, and plasma TNF-α were evaluated in all rats. RESULTS: (1) Compared with sham-EA, EA accelerated both SIT (P < 0.05) and GE (P < 0.05) and improved regularity of small intestinal slow waves. (2) Compared with the control rats (no IM), IM suppressed vagal activity and increased sympathovagal ratio assessed by the spectral analysis of heart rate variability from ECG, which were significantly prevented by EA. (3) EA significantly reduced pain score at 120 minutes (P < 0.05, vs 15 minutes) after the surgery, which was not seen with sham-EA. (4) Plasma TNF-α was increased by IM (P = 0.02) but suppressed by EA (P = 0.04) but not sham-EA. CONCLUSION: The postoperative ileus induced by IM, EA at ST36 and PC6 exerts a prokinetic effect on SIT and GE, a regulatory effect on small intestinal slow waves and an analgesic effect on postoperative pain possibly mediated via the autonomic-cytokine mechanisms.

RESUMEN

INTRODUCTION: Postoperative ileus (POI) is a heavy burden for healthcare industries and reduces the postoperative quality of life. The aim of this study was to investigate the effects and mechanisms of the intraoperative vagus nerve stimulation (iVNS) on gastrointestinal motility in a rodent model of POI. METHODS: For control group (control, n = 8), electrodes were placed on the chest wall for recording the electrocardiogram and on the stomach and small intestine for measuring gastric slow waves (GSWs) and small intestinal slow waves (SSWs). For sham group (sham, n = 8) and iVNS group (VNS, n = 8), after the same surgery as the control, intestinal manipulation (IM) was performed to induce POI. iVNS was performed during the surgery for the iVNS group. Small intestinal transit (SIT), gastric emptying (GE), postoperative pain, and plasma TNF-α were evaluated after operation. RESULTS: IM delayed GE that was normalized by iVNS (P < 0.05). iVNS reduced plasma TNF-α increased by IM (P = 0.04). iVNS prevents the injury of ileum mucosa induced by IM (P < 0.05). iVNS reduced the postoperative pain (P < 0.05). iVNS prevented the IM-induced decrease in vagal activity (sham 0-30 min vs. 150-180 min, P = 0.03, VNS 0-30 min vs. 150-180 min, P = 0.58) and increase in sympathovagal balance (sham 0-30 min vs. 150-180 min, P = 0.04, VNS 0-30 min vs. 150-180 min, P = 0.72). CONCLUSIONS: iVNS accelerates postoperative recovery by improving GE, reducing postoperative pain, and preventing the injury of ileum mucosa mediated via the autonomic mechanisms.

Asunto(s)

Vaciamiento Gástrico , Ileus/prevención & control , Recuperación de la Función , Estimulación del Nervio Vago , Animales , Modelos Animales de Enfermedad , Íleon/patología , Mucosa Intestinal/patología , Cuidados Intraoperatorios , Masculino , Dolor Postoperatorio/prevención & control , Complicaciones Posoperatorias/prevención & control , Periodo Posoperatorio , Ratas , Factor de Necrosis Tumoral alfa/sangre , Nervio Vago/fisiopatología

RESUMEN

OBJECTIVES: Clinical high-frequency spinal cord stimulation (hfSCS) (>250 Hz) applied at subperception amplitudes reduces leg and low back pain. This study investigates, via labeling for c-fos-a marker of neural activation, whether 500 Hz hfSCS applied at amplitudes above and below the dorsal column (DC) compound action potential (CAP) threshold excites dorsal horn neurons. MATERIALS AND METHODS: DC CAP thresholds in rats were determined by applying single biphasic pulses of SCS to T12 -T13 segments using pulse widths of 40 or 200 µsec via a ball electrode placed over the left DC and increasing amplitude until a short latency CAP was observed on the L5 DC and sciatic nerve. The result of this comparison allowed us to substitute sciatic nerve CAP for DC CAP. SCS at T12 -T13 was applied continuously for two hours using: sham or hfSCS at 500 Hz SCS, 40 µsec pulse width, and 50, 70, 90, or 140% CAP threshold. Spinal cord slices from T11 -L1 were immunolabeled for c-fos, and the number of c-fos-positive cells was quantified. RESULTS: 500 Hz hfSCS applied at 90 and 140% CAP threshold produced substantial (≥6 c-fos + neurons on average per slice per segment) c-fos expression in more segments between T11 and L1 than did sham stimulation (p < 0.025, 90% CAP; p < 0.001, 140% CAP, Fisher's Exact Tests) and resulted in more c-fos-positive neurons on average per slice per segment ipsilateral to than contralateral to the SCS electrode at 70, 90, and 140% CAP threshold (p < 0.01, Wilcoxon Signed Rank Tests). CONCLUSIONS: The finding of enhanced c-fos expression in the ipsilateral superficial dorsal horn provides evidence for activation/modulation of neuronal circuitry associated with subperception hfSCS.

Asunto(s)

Potenciales de Acción/fisiología , Fenómenos Biofísicos/fisiología , Modelos Animales , Células del Asta Posterior/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Estimulación de la Médula Espinal/métodos , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiología , Umbral Sensorial/fisiología

RESUMEN

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Asunto(s)

Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos , Inhibidores Enzimáticos/farmacología , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Neoplasias Pulmonares , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Nucleótido Desaminasas/antagonistas & inhibidores , Ribonucleótidos , Aminoimidazol Carboxamida/farmacocinética , Aminoimidazol Carboxamida/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Transferasas de Hidroximetilo y Formilo/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Complejos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleótido Desaminasas/metabolismo , Ribonucleótidos/farmacocinética , Ribonucleótidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Asunto(s)

Adenina/metabolismo , Bacteriófagos/fisiología , ADN Complementario/genética , ADN Polimerasa Dirigida por ARN/fisiología , Retroelementos/fisiología , Moldes Genéticos , Bordetella/virología , ADN Complementario/metabolismo , Variación Genética/efectos de los fármacos , Variación Genética/fisiología , Mutagénesis Insercional/métodos , Mutagénesis Sitio-Dirigida/métodos , Mutágenos/metabolismo , Mutágenos/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo

RESUMEN

OBJECTIVES: Spinal cord stimulation (SCS) at both conventional and higher frequencies may effectively reduce pain, but optimal parameters need to be established. This study investigated how SCS at different frequencies and pulse widths acutely modulates nociceptive activity of wide dynamic range (WDR) and high threshold (HT) dorsal horn neurons in rats at a stimulus amplitude that influences both local circuits and dorsal column fibers. MATERIALS AND METHODS: L2 -L3 and L6 -S2 spinal segments were exposed for SCS and spinal neuronal recordings, respectively. Responses to pinch of a hindpaw were recorded before and after SCS (40 or 200 µsec pulse width at 50, 500, 1 kHz and 10 kHz, amplitude: 90% of motor threshold) for 5 or 20 min. Pinch responses were tested within 30 s after SCS ceased (first pinch) and at ∼4 min intervals until response recovery. RESULTS: 1) SCS for 5 min suppressed averaged first pinch responses, except for 40 µsec/50 Hz. 2) Only SCS with 40 µs/1 kHz suppressed more spinal neurons than 200 µsec/50 Hz. 3) All SCS parameters at 5 min increased pinch responses for a small population of cells, with the incidence being greater for WDR than for HT neurons. 4) SCS at 1 kHz (40 or 200 µsec) for 20 min reduced the response to the second pinch as compared with baseline responses. In addition, no neurons exhibited increased pinch responses. CONCLUSIONS: Compared with a typical low frequency SCS (200 µs/50 Hz) or high-frequency SCS at 10 kHz, at an amplitude designed to influence both local spinal circuits and dorsal column fiber tracts, 1 kHz SCS suppressed nociceptive responses of more spinal neurons and/or demonstrated longer persisting suppressive effects. SCS at 1 kHz surpassed both low-frequency (50 Hz) and high-frequency (10 kHz) SCS application in this normal animal model.

Asunto(s)

Potenciales de Acción/fisiología , Neuronas/fisiología , Estimulación Física/efectos adversos , Estimulación de la Médula Espinal/métodos , Médula Espinal/citología , Análisis de Varianza , Animales , Biofisica , Modelos Animales de Enfermedad , Masculino , Dolor/etiología , Dolor/patología , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo