Developing a personalized closed-loop controller of medically-induced coma in a rodent model.

OBJECTIVE: Personalized automatic control of medically-induced coma, a critical multi-day therapy in the intensive care unit, could greatly benefit clinical care and further provide a novel scientific tool for investigating how the brain response to anesthetic infusion rate changes during therapy. Personalized control would require real-time tracking of inter- and intra-subject variabilities in the brain response to anesthetic infusion rate while simultaneously delivering the therapy, which has not been achieved. Current control systems for medically-induced coma require a separate offline model fitting experiment to deal with inter-subject variabilities, which would lead to therapy interruption. Removing the need for these offline interruptions could help facilitate clinical feasbility. In addition, current systems do not track intra-subject variabilities. Tracking intra-subject variabilities is essential for studying whether or how the brain response to anesthetic infusion rate changes during therapy. Further, such tracking could enhance control precison and thus help facilitate clinical feasibility. APPROACH: Here we develop a personalized closed-loop anesthetic delivery (CLAD) system in a rodent model that tracks both inter- and intra-subject variabilities in real time while simultaneously controlling the anesthetic in closed loop. We tested the CLAD in rats by administrating propofol to control the electroencephalogram (EEG) burst suppression. We first examined whether the CLAD can remove the need for offline model fitting interruption. We then used the CLAD as a tool to study whether and how the brain response to anesthetic infusion rate changes as a function of changes in the depth of medically-induced coma. Finally, we studied whether the CLAD can enhance control compared with prior systems by tracking intra-subject variabilities. MAIN RESULTS: The CLAD precisely controlled the EEG burst suppression in each rat without performing offline model fitting experiments. Further, using the CLAD, we discovered that the brain response to anesthetic infusion rate varied during control, and that these variations correlated with the depth of medically-induced coma in a consistent manner across individual rats. Finally, tracking these variations reduced control bias and error by more than 70% compared with prior systems. SIGNIFICANCE: This personalized CLAD provides a new tool to study the dynamics of brain response to anesthetic infusion rate and has significant implications for enabling clinically-feasible automatic control of medically-induced coma.

Digital Restriction Enzyme Analysis of Methylation (DREAM).

Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS) technology. The method is based on sequential cuts of genomic DNA with a pair of restriction enzymes (SmaI and XmaI) at CCCGGG target sites. Unmethylated sites are first digested with SmaI. This enzyme cuts the sites in the middle at CCC^GGG, leaving behind blunt ended fragments. CpG methylation completely blocks SmaI; therefore, only unmethylated sites are cleaved. The remaining methylated sites are digested with XmaI in the next step. This enzyme is not blocked by CpG methylation. It cuts the recognition site sideways at C^CCGGG forming 5'-CCGG overhangs. The sequential cuts thus create distinct methylation-specific signatures at the ends of restriction fragments: 5'-GGG for unmethylated CpG sites and 5'-CCGGG for methylated sites. The DNA fragments resulting from the digestions are ligated to NGS adapters. Sequencing libraries are prepared using hexanucleotide barcodes for sample identification. Individual libraries with distinct barcodes are pooled and sequenced using a paired ends protocol. The sequencing reads are aligned to the genome and mapped to unique CCCGGG target sites. Methylation at individual CpG sites is calculated as the ratio of sequencing reads with the methylated signature to the total number of reads mapping to the site. Sequencing of 25 million reads per sample typically yields 50,000 unique CpG sites covered with hundreds of reads enabling accurate determination of DNA methylation levels. DREAM does not require bisulfite conversion, has a very low background, and has high sensitivity to low levels of methylation. The method is simple, cost-effective, quantitative, highly reproducible, and can be applied to any species.

Caloric restriction delays age-related methylation drift.

In mammals, caloric restriction consistently results in extended lifespan. Epigenetic information encoded by DNA methylation is tightly regulated, but shows a striking drift associated with age that includes both gains and losses of DNA methylation at various sites. Here, we report that epigenetic drift is conserved across species and the rate of drift correlates with lifespan when comparing mice, rhesus monkeys, and humans. Twenty-two to 30-year-old rhesus monkeys exposed to 30% caloric restriction since 7-14 years of age showed attenuation of age-related methylation drift compared to ad libitum-fed controls such that their blood methylation age appeared 7 years younger than their chronologic age. Even more pronounced effects were seen in 2.7-3.2-year-old mice exposed to 40% caloric restriction starting at 0.3 years of age. The effects of caloric restriction on DNA methylation were detectable across different tissues and correlated with gene expression. We propose that epigenetic drift is a determinant of lifespan in mammals.Caloric restriction has been shown to increase lifespan in mammals. Here, the authors provide evidence that age-related methylation drift correlates with lifespan and that caloric restriction in mice and rhesus monkeys results in attenuation of age-related methylation drift.

Sevoflurane Induces Coherent Slow-Delta Oscillations in Rats.

Although general anesthetics are routinely administered to surgical patients to induce loss of consciousness, the mechanisms underlying anesthetic-induced unconsciousness are not fully understood. In rats, we characterized changes in the extradural EEG and intracranial local field potentials (LFPs) within the prefrontal cortex (PFC), parietal cortex (PC), and central thalamus (CT) in response to progressively higher doses of the inhaled anesthetic sevoflurane. During induction with a low dose of sevoflurane, beta/low gamma (12-40 Hz) power increased in the frontal EEG and PFC, PC and CT LFPs, and PFC-CT and PFC-PFC LFP beta/low gamma coherence increased. Loss of movement (LOM) coincided with an abrupt decrease in beta/low gamma PFC-CT LFP coherence. Following LOM, cortically coherent slow-delta (0.1-4 Hz) oscillations were observed in the frontal EEG and PFC, PC and CT LFPs. At higher doses of sevoflurane sufficient to induce loss of the righting reflex, coherent slow-delta oscillations were dominant in the frontal EEG and PFC, PC and CT LFPs. Dynamics similar to those observed during induction were observed as animals emerged from sevoflurane anesthesia. We conclude that the rat is a useful animal model for sevoflurane-induced EEG oscillations in humans, and that coherent slow-delta oscillations are a correlate of sevoflurane-induced behavioral arrest and loss of righting in rats.

Repositioning FDA-Approved Drugs in Combination with Epigenetic Drugs to Reprogram Colon Cancer Epigenome.

Epigenetic drugs, such as DNA methylation inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi), are approved in monotherapy for cancer treatment. These drugs reprogram gene expression profiles, reactivate tumor suppressor genes (TSG) producing cancer cell differentiation and apoptosis. Epigenetic drugs have been shown to synergize with other epigenetic drugs or various anticancer drugs. To discover new molecular entities that enhance epigenetic therapy, we performed a high-throughput screening using FDA-approved libraries in combination with DNMTi or HDACi. As a screening model, we used YB5 system, a human colon cancer cell line, which contains an epigenetically silenced CMV-GFP locus, mimicking TSG silencing in cancer. CMV-GFP reactivation is triggered by DNMTi or HDACi and responds synergistically to DNMTi/HDACi combination, which phenocopies TSG reactivation upon epigenetic therapy. GFP fluorescence was used as a quantitative readout for epigenetic activity. We discovered that 45 FDA-approved drugs (4% of all drugs tested) in our FDA-approved libraries enhanced DNMTi and HDACi activity, mainly belonging to anticancer and antiarrhythmic drug classes. Transcriptome analysis revealed that combination of decitabine (DNMTi) with the antiarrhythmic proscillaridin A produced profound gene expression reprogramming, which was associated with downregulation of 153 epigenetic regulators, including two known oncogenes in colon cancer (SYMD3 and KDM8). Also, we identified about 85 FDA-approved drugs that antagonized DNMTi and HDACi activity through cytotoxic mechanisms, suggesting detrimental drug interactions for patients undergoing epigenetic therapy. Overall, our drug screening identified new combinations of epigenetic and FDA-approved drugs, which can be rapidly implemented into clinical trials. Mol Cancer Ther; 16(2); 397-407. ©2016 AACR.

Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia.

Dopamine (DA) promotes wakefulness, and DA transporter inhibitors such as dextroamphetamine and methylphenidate are effective for increasing arousal and inducing reanimation, or active emergence from general anesthesia. DA neurons in the ventral tegmental area (VTA) are involved in reward processing, motivation, emotion, reinforcement, and cognition, but their role in regulating wakefulness is less clear. The current study was performed to test the hypothesis that selective optogenetic activation of VTA DA neurons is sufficient to induce arousal from an unconscious, anesthetized state. Floxed-inverse (FLEX)-Channelrhodopsin2 (ChR2) expression was targeted to VTA DA neurons in DA transporter (DAT)-cre mice (ChR2+ group; n = 6). Optical VTA stimulation in ChR2+ mice during continuous, steady-state general anesthesia (CSSGA) with isoflurane produced behavioral and EEG evidence of arousal and restored the righting reflex in 6/6 mice. Pretreatment with the D1 receptor antagonist SCH-23390 before optical VTA stimulation inhibited the arousal responses and restoration of righting in 6/6 ChR2+ mice. In control DAT-cre mice, the VTA was targeted with a viral vector lacking the ChR2 gene (ChR2- group; n = 5). VTA optical stimulation in ChR2- mice did not restore righting or produce EEG changes during isoflurane CSSGA in 5/5 mice. These results provide compelling evidence that selective stimulation of VTA DA neurons is sufficient to induce the transition from an anesthetized, unconscious state to an awake state, suggesting critical involvement in behavioral arousal.

Small Bowel Dose Tolerance for Stereotactic Body Radiation Therapy.

Inconsistencies permeate the literature regarding small bowel dose tolerance limits for stereotactic body radiation therapy (SBRT) treatments. In this review, we organized these diverse published limits with MD Anderson at Cooper data into a unified framework, constructing the dose-volume histogram (DVH) Risk Map, demonstrating low-risk and high-risk SBRT dose tolerance limits for small bowel. Statistical models of clinical data from 2 institutions were used to assess the safety spectrum of doses used in the exposure of the gastrointestinal tract in SBRT; 30% of the analyzed cases had vascular endothelial growth factor inhibitors (VEGFI) or other biological agents within 2 years before or after SBRT. For every dose tolerance limit in the DVH Risk Map, the probit dose-response model was used to estimate the risk level from our clinical data. Using the current literature, 21Gy to 5cc of small bowel in 3 fractions has low toxicity and is reasonably safe, with 6.5% estimated risk of grade 3 or higher complications, per Common Terminology Criteria for Adverse Events version 4.0. In the same fractionation for the same volume, if lower risk is required, 16.2Gy has an estimated risk of only 2.5%. Other volumes and fractionations are also reviewed; for all analyzed high-risk small bowel limits, the risk is 8.2% or less, and the low-risk limits have 4% or lower estimated risk. The results support current clinical practice, with some possibility for dose escalation.

Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.

Long-term results of Ingelman-Sundberg denervation procedure for urge incontinence refractory to medical therapy.

PURPOSE: Urge incontinence refractory to anticholinergic medication and behavioral techniques is a therapeutic challenge. We evaluated the durability of the modified Ingelman-Sundberg detrusor denervation procedure as minimally invasive surgical therapy for intractable urge incontinence. MATERIALS AND METHODS: Patients presenting with severe urge incontinence unresponsive to medical and/or behavioral therapy were injected subtrigonally with 10 ml. 0.25% bupivacaine. The patients were contacted 24 hours later to determine whether they experienced a decrease in urgency and urge incontinent episodes. The 28 patients with temporary resolution of symptoms were offered operative management. All patients were evaluated with history, physical examination and fluoroscopic urodynamics. The procedure consists of transvaginal dissection of the perivesical fascia from the area of the trigone, including sharp division of the terminal branches of the pelvic nerve. RESULTS: A total of 28 patients 28 to 83 years old (mean age 54.6) underwent the Ingelman-Sundberg procedure from April 1993 to September 1997. All patients presented with a history of urge incontinence, 10 reported concomitant stress incontinence and 10 had documented unstable detrusor contractions on urodynamic evaluation. Needle suspension and the pubovaginal sling procedure were performed with the Ingelman-Sundberg procedure in 1 case each. Mean followup was 44.1 months (range 14 to 67). Of the patients 15 (54%) achieved the complete durable resolution of urge incontinence, 4 (14%) were improved and 9 (32%) were unchanged. CONCLUSIONS: Ingelman-Sundberg bladder denervation resulted in a 68% long-term cure or improved rate in a difficult patient population, namely those with intractable urge incontinence. This brief, minimally invasive procedure is an excellent alternative to more aggressive surgical options.