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.

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.

Differential lung ventilation for increased oxygenation during one lung ventilation for video assisted lung surgery.

BACKGROUND: One lung ventilation (OLV) is the technique used during lung resection surgery in order to facilitate optimal surgical conditions. OLV may result in hypoxemia due to the shunt created. Several techniques are used to overcome the hypoxemia, one of which is continuous positive airway pressure (CPAP) to the non-dependent lung. Another technique is ventilating the non-dependent lung with a minimal volume, thus creating differential lung ventilation (DLV). In this study we compared the efficacy of CPAP to DLV during video assisted thoracoscopic lung resection. PATIENTS AND METHOD: This is a prospective study of 30 adult patients undergoing elective video assisted thoracoscopic lung lobectomy. Each patient was ventilated in four modes: two lung ventilation, OLV, OLV + CPAP and OLV + DLV. Fifteen patients were ventilated with CPAP first and DLV next, and the other 15 were ventilated with DLV first and then CPAP. Five minutes separated each mode, during which the non-dependent lung was open to room air. We measured the patient's arterial blood gas during each mode of ventilation. The surgeons, who were blinded to the ventilation technique, were asked to assess the surgical conditions at each stage. RESULTS: Oxygenation during OLV+ CPAP was significantly lower that OLV + DLV (p = 0.018). There were insignificant alterations of pH, PCO2 and HCO3 during the different ventilating modes. The surgeons' assessments of interference in the field exposure between OLV + CPAP or OLV + DLV was found to be insignificant (p = 0.073). CONCLUSIONS: During OLV, DLV is superior to CPAP in improving patient's oxygenation, and may be used where CPAP failed. TRIAL REGISTRATION: ClinicalTrials.gov NCT03563612 . Registered 9 June 2018, retrospectively (due to clerical error).

Pre-reproductive stress and fluoxetine treatment in rats affect offspring A-to-I RNA editing, gene expression and social behavior.

Adenosine to inosine RNA editing is an epigenetic process that entails site-specific modifications in double-stranded RNA molecules, catalyzed by adenosine deaminases acting on RNA (ADARs). Using the multiplex microfluidic PCR and deep sequencing technique, we recently showed that exposing adolescent female rats to chronic unpredictable stress before reproduction affects editing in the prefrontal cortex and amygdala of their newborn offspring, particularly at the serotonin receptor 5-HT2c (encoded by Htr2c). Here, we used the same technique to determine whether post-stress, pre-reproductive maternal treatment with fluoxetine (5 mg/kg, 7 days) reverses the effects of stress on editing. We also examined the mRNA expression of ADAR enzymes in these regions, and asked whether social behavior in adult offspring would be altered by maternal exposure to stress and/or fluoxetine. Maternal treatment with fluoxetine altered Htr2c editing in offspring amygdala at birth, enhanced the expression of Htr2c mRNA and RNA editing enzymes in the prefrontal cortex, and reversed the effects of pre-reproductive stress on Htr2c editing in this region. Furthermore, maternal fluoxetine treatment enhanced differences in editing of glutamate receptors between offspring of control and stress-exposed rats, and led to enhanced social preference in adult offspring. Our findings indicate that pre-gestational fluoxetine treatment affects patterns of RNA editing and editing enzyme expression in neonatal offspring brain in a region-specific manner, in interaction with pre-reproductive stress. Overall, these findings imply that fluoxetine treatment affects serotonergic signaling in offspring brain even when treatment is discontinued before gestation, and its effects may depend upon prior exposure to stress.

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.

The effect of 'Out of hours surgery Service' in Israel on hip fracture fixation outcomes: a retrospective analysis.

BACKGROUND: 'Out of Hours Surgery Service' (OHSS) was implemented in Israel, amongst other reasons, in order to reduce the time interval between hospital admission and surgery and consequently improve outcomes. The OHSS is currently operated in the public hospitals in Israel. In this study we compared the data of patients before and after OHSS implementation to determine its efficacy in improving patient care. METHODS: This is a retrospective observational study of 792 adult patients who underwent hip fracture surgery between 2002 and 2007 in a single hospital. The study population included two groups: patients that were operated before the implementation of the OHSS (2002-2004) and after the implementation of the OHSS (2005-2007). Data regarding all patients was collected using the institution's computer program. The following variables were analyzed: patients' demographics, time interval from hospitalization to surgery, causes for delaying surgery, post-operative length of hospitalization and mortality. RESULTS: Patients in the post-OHSS group had more illnesses and higher ASA classification than those in the pre-OHSS group. The post-OHSS group had a significantly decreased length of stay in the hospital before and after the surgery. After adjusting for ASA score and age, the post-OHSS group was found to have decreased post-operative hospitalization and lower post-operative mortality. Surgery was delayed in pre-OHSS period mainly due to operating rooms unavailability. CONCLUSION: Implementation of OHSS facilitated operating room availability, thus early operation and reduced post-operative mortality. In accordance with other studies, patient's outcome is greatly influenced by the time from admission to hip fracture surgery.

Abnormalities in A-to-I RNA editing patterns in CNS injuries correlate with dynamic changes in cell type composition.

Adenosine to Inosine (A-to-I) RNA editing is a co- or post-transcriptional mechanism that modifies genomically encoded nucleotides at the RNA level. A-to-I RNA editing is abundant in the brain, and altered editing levels have been reported in various neurological pathologies and following spinal cord injury (SCI). The prevailing concept is that the RNA editing process itself is dysregulated by brain pathologies. Here we analyzed recent RNA-seq data, and found that, except for few mammalian conserved editing sites, editing is significantly higher in neurons than in other cell populations of the brain. We studied A-to-I RNA editing in stab wound injury (SWI) and SCI models and showed that the apparent under-editing observed after injury correlates with an approximately 20% reduction in the relative density of neurons, due to cell death and immune cell infiltration that may account for the observed under-editing. Studies of neuronal and astrocyte cultures and a computational analysis of SCI RNA-seq data further supported the possibility that a reduction in neuronal density is responsible for alterations in the tissue-wide editing patterns upon injury. Thus, our data suggest that the case for a mechanistic linkage between A-to-I RNA editing and brain pathologies should be revisited.

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.

Expanding preconception carrier screening for the Jewish population using high throughput microfluidics technology and next generation sequencing.

BACKGROUND: Genetic screening to identify carriers of autosomal recessive diseases has become an integral part of routine prenatal care. In spite of the rapid growth of known mutations, most current screening programs include only a small subset of these mutations, and are performed using diverse molecular techniques, which are generally labor-intensive and time consuming. We examine the implementation of the combined high-throughput technologies of specific target amplification and next generation sequencing (NGS), for expanding the carrier screening program in the Israeli Jewish population as a test case. METHODS: We compiled a panel of 370 germline mutations, causing 120 disorders, previously identified in affected Jewish individuals from different ethnicities. This mutation panel was simultaneously captured in 48 samples using a multiplex PCR-based microfluidics approach followed by NGS, thereby performing 17,760 individual assays in a single experiment. RESULTS: The sensitivity (measured with depth of at least 50×) and specificity of the target capture was 98 and 95 % respectively, leaving minimal rate of inconclusive tests per sample tested. 97 % of the targeted mutations present in the samples were correctly identified and validated. CONCLUSION: Our methodology was shown to successfully combine multiplexing of target specific primers, samples indexing and NGS technology for population genetic screens. Moreover, it's relatively ease of use and flexibility of updating the targets screened, makes it highly suitable for clinical implementation. This protocol was demonstrated in pre-conceptional screening for pan-Jewish individuals, but can be applied to any other population or different sets of mutations.

Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis.

In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection.

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).

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.

Airway Management of the Patient with Maxillofacial Trauma: Review of the Literature and Suggested Clinical Approach.

According to the Advanced Trauma Life Support recommendations for managing patients with life-threatening injuries, securing the airway is the first task of a primary caregiver. Airway management of patients with maxillofacial trauma is complex and crucial because it can dictate a patient's survival. Securing the airway of patients with maxillofacial trauma is often extremely difficult because the trauma involves the patient's airway and their breathing is compromised. In these patients, mask ventilation and endotracheal intubation are anticipated to be difficult. Additionally, some of these patients may not yet have been cleared of a cervical spine injury, and all are regarded as having a full stomach and having an increased risk of regurgitation and pulmonary aspiration. The requirements of the intended maxillofacial operation may often preclude the use of an oral intubation tube, and alternative methods for securing the airway should be considered before the start of the surgery. In order to improve the clinical outcome of patients with maxillofacial trauma, cooperation between maxillofacial surgeons, anesthesiologists, and trauma specialists is needed. In this review, we discuss the complexity and difficulties of securing the airway of patients with maxillofacial trauma and present our approach for airway management of such patients.

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.

Subcutaneous Emphysema following Emergent Surgical Conventional Tracheostomy.

In maxillofacial surgery, tracheostomy is indicated in congenital, inflammatory, oncologic, or traumatic respiratory obstruction. In traumatic cases, however, it is sometimes hard to implement. We describe subcutaneous emphysema following emergent surgical conventional tracheostomy performed after stab injury to the floor of the mouth. We analyze the course that led to this complication and discuss suggestions on how to avoid it. In addition, we review the literature to improve our knowledge and practice regarding this entity. Massive subcutaneous neck emphysema occurred because ventilation started at the time when the hemorrhage was not completely managed and the tracheal tube was not fully secured. In traumatic cases with profound bleeding, hemorrhage management must be performed carefully. The recommendation not to ventilate until the hemorrhage is completely managed should be observed.

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.

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.