A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels. RESULTS: In agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes. CONCLUSIONS: Our findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.

Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease.

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients' brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.

Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish.

Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the fmr1 zebrafish mutant (fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS.

A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes.

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Indications for Elective Tracheostomy in Reconstructive Surgery in Patients With Oral Cancer.

BACKGROUND: Oral cancer surgery carries a high risk of upper airway obstruction; yet optimal airway management approach remains controversial. AIM OF STUDY: The purpose of the present study was to evaluate the use of tracheostomy in oncological patients undergoing oral cancer surgery with intra oral flap reconstruction. METHODS: The study cohort included 75 patients with oral cancer, who underwent major intraoral resections and reconstruction with vascularized flaps. RESULTS: Thirty-six percent of the patients received elective tracheostomy (27 patients). Mean hospital stay of the patients with tracheostomy was 28.4 ±â€Š12.5 days compared with 9.7 ±â€Š2.1 days in the nontracheostomy patients. A scoring system rendered from this study suggests that patients with a total scoring at or above 8 should be considered for elective tracheostomy. CONCLUSIONS: With appropriate postoperative monitoring, selected patients can be managed without routine elective tracheostomy, yet, patients with comorbidities, mostly elderly patients, which undergo surgical resection and reconstruction in high-risk areas that can result in a bulky flap that pose danger to the postoperative airway, should receive elective tracheostomy.

The Role of Fine Needle Aspiration in the Diagnosis of Parotid Gland Tumors: Correlation With Preoperative Computerized Tomography Tumor Size.

The role of fine needle aspiration cytology (FNAC) in the diagnosis of parotid gland masses is still controversial, regarding its sensitivity and specificity that vary between 41% and 100% and between 86% and 100% respectively.The aim of this study was to identify the specificity and sensitivity of FNAC of parotid gland tumors in relation to the tumor size as characterized preoperatively by computer tomography. The medical files of 79 patients whom were referred to the MaxilloFacila Surgery Department, Rambam medical center, over a 10.5-year period (2000-2010) were analyzed retrospectively.The extensity of the operation was determined by the location of the tumor as presented in computed tomography (CT) radiography, and preoperative FNAC examination.The majority of the masses were located in the superficial lobe (88.52%), and only 11.48% of the patients were located in the deep lobe (8:1 ratio). FNAC results were nondiagnostic in 7 patients (8.86%), 62 patients were diagnosed as inflammatory and benign lesion in (78.48%), malignant tumors were diagnosed in 10 patients (12.65%).The sensitivity in our study was 90%, the specificity was 98%, positive predictive value was 90%, negative predictive value was 98%, and diagnostic accuracy was 88%. The positive predictive value was 90%, the negative predictive value was 98%.Analyzing the effect of the preoperative CT size upon the accuracy of the FNAC diagnosis, we found that lesion with preoperative CT size greater than 24 mm has a more accurate FNAC result (P = 0.034).

Hypotensive anesthesia versus normotensive anesthesia during major maxillofacial surgery: a review of the literature.

Steady blood pressure within normal limits during surgery is one of the markers of the ideal and skillful anesthesia. Yet, reduced blood pressure is advantageous in some settings because it can contribute to a reduction in overall blood loss and improve the surgical field conditions. Controlled hypotension during anesthesia or hypotensive anesthesia is often used in major maxillofacial operations. Since hypotensive anesthesia carries the risk of hypoperfusion to important organs and tissues, mainly the brain, heart, and kidneys, it cannot be applied safely in all patients. In this paper we review the medical literature regarding hypotensive anesthesia during major maxillofacial surgery, the means to achieve it, and the risks and benefits of this technique, in comparison to normotensive anesthesia.

The use of VivaSight™ single lumen endotracheal tube in morbidly obese patients undergoing laparoscopic sleeve gastrectomy.

BACKGROUND: The population of obese patients is progressively growing and bariatric operations are becoming increasingly common. Morbidly obese patients require special anesthetic care and are often considered to be difficult to ventilate and intubate. The VivaSight™ Single Lumen tube is an endotracheal tube with a camera embedded in its tip. The view from the tip appears continuously on a monitor in the anesthesiologist's vicinity. The aim of this study was to assess the VivaSight™ in comparison with conventional endotracheal tube as an aid in the intubation and surveillance of tube position during surgery of obese patients. METHODS: This is a prospective study of 72 adult obese patients who underwent laparoscopic sleeve gastrectomy. The patients were randomly assigned to be intubated by either the VivaSight™ (40 patients, test group) or a conventional endotracheal tube (32 patients, control group). Data on the patients, the pre-operative airway evaluation, the endotracheal intubation and the post-operative outcome were collected and compared. RESULTS: The Mallampati scores were significantly higher in the test group than in the control group. Endotracheal intubation took 29 ± 10 and 24 ± 8 seconds using the VivaSight™ and a conventional tube respectively (p = 0.02). Three of the patients in the control group, while none of those in the test group, had soft tissue injury (p < 0.05). CONCLUSION: We found the VivaSight™ SL to be helpful in the endotracheal intubation and continuous surveillance of tube position in morbidly obese patients undergoing laparoscopic sleeve gastrectomy.

Core decompression and alendronate treatment of the osteonecrotic rat femoral head: computer-assisted analysis.

Femoral head avascular necrosis is a process leading to femoral head deformity and osteoarthritic changes in the hip joint. Alendronate slows down bone resorption and remodelling in rats, while core decompression hastens the healing processes. We evaluated the influence of daily alendronate treatment on the rat femoral head shape after surgical osteonecrosis with core decompression, compared with controls. No differences were found in shape factor and femoral head height/length ratios. It was concluded that alendronate treatment slows down the process of replacing osteonecrotic bone by new bone and prevents early immature new bone collapse resulting from early revascularization because of core decompression.

Factors associated with adverse outcome following urologic surgery in patients aged 80 years and older.

BACKGROUND AND AIMS: This study was designed to find predictors for adverse post-operative outcomes in elderly patients aged 80 years and older, who underwent urologic surgery. METHODS: In this prospective observational study, we analysed data, including age, gender, American Society of Anesthesiologists (ASA) class, co-morbidities, number of regular medications, type and extent of surgery, type of anesthesia, duration of surgery and hospitalization, post-operative morbidity and mortality. We studied the correlations between each pre- and intra-operative parameter to the consequence, to find predictors for adverse outcome. RESULTS: During a 12-month period, 217 patients underwent 294 urologic procedures in our institution. Ninety-eight procedures (33%) were followed by complications and 11 patients (5%) died. Patients who had uneventful surgery and hospitalization were significantly younger than those who experienced morbidity or mortality. There was a significantly higher complication rate among patients with a higher ASA class or with ischemic heart disease, following higher graded or longer operations, and after emergency surgery. CONCLUSIONS: Older age is a significant risk factor. Patients with higher ASA class or ischemic heart disease are at higher risk for post-operative complications and thus, require careful follow-up. In this age population, extensive or prolonged surgery should be carefully considered.

Ig gene diversification and selection in follicular lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma revealed by lineage tree and mutation analyses.

Follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL) and primary central nervous system lymphoma are B cell malignancies. FL and DLBCL have a germinal center origin. We have applied mutational analyses and a novel algorithm for quantifying shape properties of mutational lineage trees to investigate the nature of the diversification, somatic hypermutation and selection processes that affect B cell clones in these malignancies and reveal whether they differ from normal responses. Lineage tree analysis demonstrated higher diversification and mutations per cell in the lymphoma clones. This was caused solely by the longer diversification times of the malignant clones, as their recent diversification processes were similar to those of normal responses, implying similar mutation frequencies. Since previous analyses of antigen-driven selection were shown to yield false positives, we performed a corrected analysis of replacement and silent mutation patterns, which revealed selection against replacement mutations in the framework regions, responsible for the structural integrity of the B cell receptor, but not for positive selection for replacements in the complementary determining regions. Most replacements, however, were neutral or conservative, suggesting that if at all selection operates in these malignancies it is for structural B cell receptor integrity but not for antigen binding.

Diabetes blockade of sevoflurane postconditioning is not restored by insulin in the rat heart: phosphorylated signal transducer and activator of transcription 3- and phosphatidylinositol 3-kinase-mediated inhibition.

BACKGROUND: The possibility of restoring sevoflurane postconditioning (sevo-postC) cardioprotection in diabetic animals is uncertain. We hypothesized that attenuation of myocardial injury by sevo-postC might be hindered by inhibition of signal transducer and activator of transcription (STAT) 3-regulated activity of phosphatidylinositol 3-kinase (PI3K) in diabetic animals. To determine whether postC cardioprotection can be restored by normoglycemia, we treated rats with insulin. METHODS: Diabetic or nondiabetic rats were randomly subjected to 30-min ischemia/reperfusion, with ischemic postC or sevo-postC, with and without mitochondrial adenosine triphosphate-dependent potassium channel blocker 5-hydroxy decanoate sodium and PI3K antagonist wortmannin. The infarct area, phosphorylated STAT3, and apoptosis were examined. Studies were repeated after insulin treatment. RESULTS: Ischemic postC and sevo-postC significantly reduced infarct size by 50% in the nondiabetic rats (P < 0.002), a phenomenon completely reversed by 5-hydroxy decanoate sodium and wortmannin. Diabetes mellitus blocked the protective effect of postC, and insulin treatment to achieve normoglycemia did not restore cardioprotection. Phosphorylated STAT3 nuclear retention was significantly increased after ischemia-reperfusion and was further enhanced in response to ischemic postC (P < 0.05) but was significantly reduced in diabetic rats (by 43%; P < 0.01). CONCLUSIONS: The effective reduction in infarct size and apoptosis in the nondiabetic rat heart by postC was completely abrogated in diabetic rats. This inhibition is not relieved by insulin-induced normoglycemia. The PI3K pathway and mitochondrial adenosine triphosphate-dependent potassium channel activation are involved in the mechanism of postC. In diabetic rats, STAT3 activation was strongly reduced, as was postC cardioprotection, suggesting that the inability of insulin to restore postC may be attributed to diabetes-induced STAT3-mediated inhibition of PI3K signaling.

Evidence for large diversity in the human transcriptome created by Alu RNA editing.

Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome.

[Anesthesia and peri-operative care of the elderly patient].

With raised life expectancy, there is a constant elevation in the percentage of elderly people in the general population. Consequently, the age of the surgical patient increases. This progressive process results in operating on patients who were considered 'too old for surgery' a decade ago. There are many physiological and pathological changes that take place in all body systems during aging. The function capacity of organs deteriorates, resulting in decreased capability to overcome surgical stress. As a result, the elderly surgical patients have higher rates of peri-operative morbidity and mortality. Anesthesia and peri-operative care should be customized to this population. Practicing management of older patients undergoing surgery will increase the experience of all caregivers and, in time, improve outcome.

RNA editing of the 5-HT2C receptor in the central nucleus of the amygdala is involved in resilience behavior.

Post-traumatic-stress-disorder (PTSD) is a stress-related condition that may develop after exposure to a severe trauma-event. One of the core brain areas that is considered to be a key regulatory region of PTSD is the amygdala. Specifically, the central amygdala (CeA) is involved in emotion processing and associative fear learning memory, two main circuits involved in PTSD. Long term dysregulation of trauma-related emotional processing may be caused by neuroadaptations that affect gene expression. The adenosine-(A) to-inosine (I) RNA editing machinery is a post-transcriptional process that converts a genomic encoded A to I and is critical for normal brain function and development. Such editing has the potential to increase the transcriptome diversity, and disruption of this process has been linked to various central nervous system disorders. Here, we employed a unique animal model to examine the possibility that the RNA editing machinery is involved in PTSD. Detection of RNA editing specifically in the CeA revealed changes in the editing pattern of the 5-HT2C serotonin receptor (5-HT2CR) transcript accompanied by dynamic changes in the expression levels of the ADAR family enzymes (ADAR and ADARb1). Deamination by ADAR and ADARb1 enzymes induces conformational changes in the 5-HT2CR that decrease the G-protein-coupling activity, agonist affinity, and thus serotonin signaling. Significantly, a single intra-CeA administration of a 5-HT2CR pharmacological antagonist produced a robust alleviation of PTSD-like behaviors (that was maintained for three weeks) as well as single systemic treatment. This work may suggest the way to a new avenue in the understanding of PTSD regulation.

Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases.

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases. A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ, in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes. However, it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs. The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion, mutation and selection in GC reactions. We have applied several mutation analysis methods, based on lineage tree construction, to a large set of data, containing IgV productive and non-productive heavy and light chain sequences from several different tissues, to examine three of the most profoundly studied AI diseases - Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Sjögren's Syndrome (SS). We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs, but a stricter selection compared to normal controls, which was more apparent in RA. SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection, also showing differences between light and heavy chain IgV and between different tissues. The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed, relative to those in normal secondary lymphoid tissues.

Purifying selection of long dsRNA is the first line of defense against false activation of innate immunity.

BACKGROUND: Mobile elements comprise a large fraction of metazoan genomes. Accumulation of mobile elements is bound to produce multiple putative double-stranded RNA (dsRNA) structures within the transcriptome. These endogenous dsRNA structures resemble viral RNA and may trigger false activation of the innate immune response, leading to severe damage to the host cell. Adenosine to inosine (A-to-I) RNA editing is a common post-transcriptional modification, abundant within repetitive elements of all metazoans. It was recently shown that a key function of A-to-I RNA editing by ADAR1 is to suppress the immunogenic response by endogenous dsRNAs. RESULTS: Here, we analyze the transcriptomes of dozens of species across the Metazoa and identify a strong genomic selection against endogenous dsRNAs, resulting in their purification from the canonical transcriptome. This purifying selection is especially strong for long and nearly perfect dsRNAs. These are almost absent from mRNAs, but not pre-mRNAs, supporting the notion of selection due to cytoplasmic processes. The few long and nearly perfect structures found in human transcripts are weakly expressed and often heavily edited. CONCLUSION: Purifying selection of long dsRNA is an important defense mechanism against false activation of innate immunity. This newly identified principle governs the integration of mobile elements into the genome, a major driving force of genome evolution. Furthermore, we find that most ADAR1 activity is not required to prevent an immune response to endogenous dsRNAs. The critical targets of ADAR1 editing are, likely, to be found mostly in non-canonical transcripts.

Posterior reversible encephalopathy syndrome after combined general and spinal anesthesia with intrathecal morphine.

We describe a patient who was in a stupor for several days after combined general-spinal anesthesia. Both clinical manifestations and magnetic resonance imaging findings were consistent with posterior reversible encephalopathy syndrome and resolved after the patient recovered spontaneously.