CDR grafting and site-directed mutagenesis approach for the generation and affinity maturation of Anti-CD20 nanobody.

BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment. METHODS AND RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20. CONCLUSION: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.

Comparison of the effectiveness of online supportive parenting intervention (SPACE) and timid to tiger program (FTTT) on childhood anxiety disorders and family accommodation with samples of Iranian parents.

Studies have supported the effectiveness of the From Timid to a Tiger (FTTT) and Supportive Parenting for Anxious Childhood Emotions (SPACE) program in reducing childhood anxiety. This study is the first to compare the effectiveness of the two programs in the treatment of childhood anxiety disorder and reducing family accommodations levels. Parents of children aged 6 to 9 (n = 49, 49% boys) were randomly allocated to either FTTT (n 26) or SPACE (n = 23) groups, and each attended ten online sessions following the manuals of the interventions. Throughout the study, 9 participants dropped out, resulting in a total of 49 participants, and we performed statistical analyses based on data from these 49 participants. The assessment took place pre- and post-treatment and at a ten-week follow-up on parent-rated child anxiety and family accommodation measures. Analyses of variance (repeated measures) and gain scores were conducted to examine the data. The results indicated that both treatments approaches produced significant reductions in outcome measures, and the post-treatment gains of both treatments were maintained at a ten-week follow-up, though when considering the mean differences and effect sizes across the assessment scores and between groups, overall, the FTTT was significantly more effective in reducing child anxiety scores and family accommodation levels. Our results demonstrated that FTTT significantly outperformed the SPACE program in reducing childhood anxiety problems and family accommodation levels.

Investigation of a group of Iranian theater artists' mental health and attitude toward patients with mental disorders.

Introduction: Stigmatizing attitude toward patients with severe mental disorders is one of the main obstacles of improving the mental health of societies. Media plays an important role in how the public views mental health issues. Thus, we have performed this study to investigate the Iranian theater artists' mental health status, and their view toward patients with severe mental disorders. Methods: This cross-sectional study was performed via an online anonymous survey including the Social Distance Scale and the Dangerousness Scale measuring the attitude of participants toward patients with severe mental disorders, and the 28-item General Health Questionnaire (GHQ-28). It was disseminated to artists who had the experience of working in theater in the past year in Iran. Results: Our survey was responded by 104 artists. Social Distance Scale scores' mean was 10.67 (scores can range from 0 to 21) and the Dangerousness Scale scores' mean was 28.87 (scores can range from 8 to 56); higher scores indicate worse discrimination. Our participants' strongest fears were to let someone with a severe mental disorder to take care of their children, and for these groups of patients to obtain a hunting license. Twenty-six (25%) participants were at risk of moderate to severe anxiety, and 18 (17.3%) participants were at risk of moderate to severe depression. Conclusion: By and large, our participants did not have a positive attitude toward patients with severe mental disorders. Providing the knowledge of mental health issues can help the general public to be more tolerant of the mentally ill and specifically, theater can be employed to fight stigmatizing mental health issues by educating its audience.

Cilostazol as an adjunctive treatment in major depressive disorder: a pilot randomized, double-blind, and placebo-controlled clinical trial.

BACKGROUND: Cilostazol, a phosphodiesterase-3 inhibitor, has been reported to improve depressive-like behavior in experimental studies of depression. We investigated the safety and efficacy of cilostazol combination therapy with sertraline in treating patients with major depressive disorder (MDD) in a 6-week, parallel, randomized controlled trial. METHOD: Among patients referred to the outpatient clinic of a tertiary hospital, those with a diagnosis of MDD with moderate to severe severity (a score of >19 on the Hamilton depression rating scale (HAM-D)) were enrolled. A total of 54 MDD patients aged 18-65 years were randomly assigned to either the cilostazol (100 mg daily) or the placebo group. Both groups received sertraline 100 mg per day similarly. Changes in HAM-D at weeks 2, 4, and 6 were the primary outcome. Participants and outcome assessors were blinded. RESULTS: At week 6, patients in the cilostazol group had significantly lower HAM-D score (p value= 0.015). General linear model repeated-measure analysis showed significant effect for treatment in improving MDD severity (p value <0.001). The remission rate at the study endpoint and number of responders at week 4 were significantly higher in the cilostazol group (p value= 0.047, p value= 0.032, respectively). The cilostazol group demonstrated a significantly shorter time to response. No significant difference was observed in treatment response at the study endpoint, and there were no serious adverse effects. CONCLUSION: Our study supports safety and efficacy of cilostazol in treating MDD patients. TRIAL REGISTRATION: This trial was registered at the Iranian registry of clinical trials (IRCT: www.irct.ir ; registration number: IRCT20090117001556N130).

Evaluating a new verbal working memory-balance program: a double-blind, randomized controlled trial study on Iranian children with dyslexia.

BACKGROUND: It is important to improve verbal Working Memory (WM) in reading disability, as it is a key factor in learning. There are commercial verbal WM training programs, which have some short-term effects only on the verbal WM capacity, not reading. However, because of some weaknesses in current verbal WM training programs, researchers suggested designing and developing newly structured programs that particularly target educational functions such as reading skills. In the current double-blind randomized clinical trial study, we designed a new Verbal Working Memory-Balance (VWM-B) program which was carried out using a portable robotic device. The short-term effects of the VWM-B program, on verbal WM capacity, reading skills, and postural control were investigated in Iranian children with developmental dyslexia. RESULTS: The effectiveness of the VWM-B program was compared with the VWM-program as a traditional verbal WM training. In comparison with VWM-program, the participants who received training by the VWM-B program showed superior performance on verbal WM capacity, reading skills, and postural control after a short-term intervention. CONCLUSIONS: We proposed that the automatized postural control resulting from VWM-B training had a positive impact on improving verbal WM capacity and reading ability. Based on the critical role of the cerebellum in automatizing skills, our findings support the cerebellar deficit theory in dyslexia. TRIAL REGISTRATION: This trial was (retrospectively) registered on 8 February 2018 with the Iranian Registry of Clinical Trials (IRCT20171219037953N1).

MODELING FATE AND TRANSPORT OF VOLATILE ORGANIC COMPOUNDS (VOCs) INSIDE SEWER SYSTEMS.

Hazardous waste site investigations have shown that volatile organic compounds (VOCs) can be transported via sewer pipes and migrate into indoor spaces. Despite field data confirming the presence of this exposure pathway, there is lack of context-based numerical models that provide guidance to characterize and predict VOCs concentration in sewer gas at vapor intrusion sites. Particularly, this poses a challenge when assessing and mitigating risks associated with these exposure pathways. Therefore, a numerical model has been developed to simulate the concentration of VOCs in sewer gas in different stages throughout the sewer lines. The developed model considers various input parameters, including temperature, sewer liquid depth, groundwater depth, and sewer construction characteristics to incorporate local and operational conditions. The model's output is verified using field data from a sewer system constructed near a Superfund site. Moreover, a sensitivity analysis was conducted to evaluate the model's response to variation of the external input parameters. To the best of our knowledge, this study is the first attempt to model VOCs concentration in sewer gas, particularly to address vapor intrusion. The developed model can be used as a numerical tool to support the development of sewer assessment guidelines, risk assessment studies, and mitigation strategies.

Comparison of Auricular Therapy with Sham in Children with Attention Deficit/Hyperactivity Disorder: A Randomized Controlled Trial.

Objectives: Several studies have shown the clinical effects of auricular therapy for some neuropsychological disorders. The aim of this study is to compare the effectiveness of auricular therapy with the sham procedure in attention deficit hyperactivity disorder (ADHD). Design: Randomized sham-controlled trial. Settings/Location: Welfare Centers of Tehran (Iran). Subjects: Fifty children with ADHD (6-14 years old). Interventions: Patients were randomly assigned into two groups to receive either auricular therapy (Group A) or a sham procedure (Group B) once a week for 6 weeks. Outcome measures: The Children Symptom Inventory (CSI-4) and the parent's version of the Conners Comprehensive Behavior Rating Scale were used to assess the severity of symptoms of attention deficit (AD) and hyperactivity (HA). Outcome evaluation data was obtained at the first and seventh weeks after the interventions. Data were analyzed by SPSS software using Friedman and Mann-Whitney U tests. Results: There were 23 patients in group A and 21 patients in group B who completed the study. Based on the CSI-4 assessment, AD scores decreased from the mean (±standard deviation) of 18.39 (±5.44) to 15.39 (±5.89), p = 0.006 in group A, whereas the mean AD scores for group B only changed from 15.0 (±6.4) to 14.9 (±5.94), p = 0.55 in group B. In addition, the mean of the HA scores decreased from 18.0 (±6.73) to 13.3 (±6.75), p = 0.001 in group A, whereas the change in HA scores in group B only diminished and from 11.85 (±6.44) to 11.45 (±5.44), p = 0.74. The CSI-4 assessments and the scores on the Conners questionnaire significantly decreased after the first week of intervention (p = 0.04) in group A, but not in group B. No side effects were observed. Conclusion: Subjects who received auricular therapy in acupuncture points achieved a statistically significant greater reduction in AD and HA symptoms when compared with subjects who received sham acupuncture points.

Comparison of modeled and measured indoor air trichloroethene (TCE) concentrations at a vapor intrusion site: influence of wind, temperature, and building characteristics.

There is a lack of vapor intrusion (VI) models that reliably account for weather conditions and building characteristics, especially at sites where active alternative pathways, such as sewer connections and other preferential pathways, are present. Here, a method is presented to incorporate freely-available models, CONTAM, and CFD0, to estimate site-specific building air exchange rates (AERs) and indoor air contaminant concentrations by accounting for weather conditions and building characteristics at a well-known VI site with a land drain preferential pathway. To account for uncertainty in model input parameters that influence indoor air chlorinated volatile organic compound (CVOC) concentration variability, this research incorporated Monte Carlo simulations and compared model results with retrospective field data collected over approximately 1.5 years from the study site. The results of this research show that mass entry rates for TCE are likely influenced by indoor air pressures that can be modeled as a function of weather conditions (over seasons) and building characteristics. In addition, the results suggest that temporal variability in indoor air TCE concentrations is greatest (modeled and measured) due to the existence of a land drain, which acts as a preferential pathway, from the subsurface to the granular fill beneath the floor slab. The field data and modeling results are in good agreement and provide a rare comparison of field data and modeling results for a VI site. The modeling approach presented here offers a useful tool for decision makers and VI practitioners as they assess these complex and variable processes that have not been incorporated within other VI models.

L-Carnosine combination therapy for major depressive disorder: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Evidence for antidepressant effects of L-Carnosine was shown in some experimental studies. In this study we tried to evaluate the efficacy and tolerability of L-Carnosine combination therapy in treatment of patients with major depressive disorder (MDD). METHODS: Fifty-eight patients with MDD (DSM-V) and Hamilton Depression Rating Scale (HAM-D) score ≥ 19 were randomized to receive either 400 mg twice daily L-Carnosine or placebo in addition to citalopram (maximum dosage of 40 mg/day) for six weeks in a randomized double-blind, and placebo-controlled study. Patients were assessed using the HAM-D scale at baseline and weeks 2, 4, and 6. RESULTS: Fifty-two patients completed the trial. General linear model repeated measure showed significant difference for time × treatment on HAM-D score [F = 3.17, df = 2.39, p-value = 0.03]. Significantly greater improvement was detected in HAM-D score of the L-Carnosine group compared with the placebo group from baseline to weeks 2, 4 and 6 [Ps = 0.013, 0.028 and 0.023; respectively]. Patients in the L-Carnosine group experienced significantly greater response and remission rate than the placebo group [Ps = 0.023 and 0.012; respectively]. There was no significant difference between the two groups in baseline parameters and frequency of side effects. LIMITATIONS: Short follow-up period and small population size were two important limitations of this study. CONCLUSIONS: L-Carnosine combination therapy with citalopram can effectively improve symptoms of patients with major depressive disorder. Rapid-onset antidepressant effects of L-Carnosine were also shown which need further investigation.

Fluvoxamine combination therapy with tropisetron for obsessive-compulsive disorder patients: A placebo-controlled, randomized clinical trial.

BACKGROUND: About 50% of obsessive-compulsive disorder patients still suffer significant symptoms even after the recommended first-line therapy. This demonstrates the necessity to investigate strategies to improve alleviation of symptoms. OBJECTIVE: The main objective of this study was to investigate the efficacy of a 5-hydroxytryptophan 3 receptor antagonist, tropisetron, as an adjuvant therapy to selective serotonin reuptake inhibitors, in ameliorating obsessive-compulsive disorder symptoms. METHODS: Men and women between the ages of 18-60 years diagnosed with obsessive-compulsive disorder, based on DSM5, who had a Yale-Brown obsessive compulsive scale score of more than 21 were recruited in a double-blinded, parallel-group, placebo-controlled, clinical trial of 10 weeks to receive either tropisetron (5 mg twice daily) and fluvoxamine (100 mg daily initially followed by 200 mg daily after week 4) or placebo and fluvoxamine. The primary outcome of interest in this study was the Yale-Brown obsessive compulsive scale total score decrease from baseline. RESULTS: One hundred and eight participants were equally randomized into two groups; 48 participants in each group finished the trial. The Yale-Brown obsessive compulsive total score significantly dropped in both groups while the tropisetron group participants experienced a significantly higher decrease in their scores (Greenhouse-Geisser F(1.53-65.87)=3.516, p-value=0.04). No major adverse effect was observed in any of the groups. CONCLUSION: This trial showed a significant efficacy for tropisetron over placebo in treatment of obsessive-compulsive disorder symptoms when added to fluvoxamine.

Building science approaches for vapor intrusion studies.

Indoor air concentrations are susceptible to temporal and spatial variations and have long posed a challenge to characterize for vapor intrusion scientists, in part, because there was a lack of evidence to draw conclusions about the role that building and weather conditions played in altering vapor intrusion exposure risks. Importantly, a large body of evidence is available within the building science discipline that provides information to support vapor intrusion scientists in drawing connections about fate and transport processes that influence exposure risks. Modeling tools developed within the building sciences provide evidence of reported temporal and spatial variation of indoor air contaminant concentrations. In addition, these modeling tools can be useful by calculating building air exchange rates (AERs) using building specific features. Combining building science models with vapor intrusion models, new insight to facilitate decision-making by estimating indoor air concentrations and building ventilation conditions under various conditions can be gained. This review highlights existing building science research and summarizes the utility of building science models to improve vapor intrusion exposure risk assessments.

Simultaneous removal of a cationic and an anionic textile dye from water by a mixed sorbent of vermicompost and Persian charred dolomite.

The potential of a mixed sorbent consisting of vermicompost and Persian charred dolomite for simultaneous adsorption of Basic Violet 16 (BV16) and Reactive Red 195 (RR195) was investigated. First-order derivative spectrophotometry was used for simultaneous analysis of the two dyes. In single dye experiments, the maximum adsorption capacity of vermicompost for BV16 was found to be 16 mg g-1 and the adsorption capacity of charred dolomite for RR195 was 7.3 mg g-1. Anionic RR195 was not noticeably adsorbed by vermicompost (negative surface charge) and cationic BV16 not by charred dolomite (positive surface charge) but adsorbed by the oppositely charged adsorbents which indicates a selective electrostatic adsorption mechanism. In binary dye solution, BV16 adsorption onto charred dolomite was increased in the presence of RR195 (synergistic effect), yet RR195 adsorption on charred dolomite was not influenced by BV16. An antagonistic effect of RR195 was concluded for BV16 adsorption onto vermicompost. The adsorption equilibrium data for both adsorbents fitted more acceptable to the Langmuir isotherm model than to the Freundlich model in single and binary solutions, but other than the adsorption of BV16 on vermicompost in binary solution which followed the Freundlich model. More than 50% of the removal efficiencies determined for both dyes onto the mixed sorbents were >70% which highlights that the mixed sorbent investigated is highly efficacious for the simultaneous removal of cationic and anionic dyes from contaminated groundwater. Eight cycles reusing vermicompost with 1 N NaOH for regeneration demonstrates the practicability and economic advantage of this natural biosorbent.

Postural Balance Performance of Children with ADHD, with and without Medication: A Quantitative Approach.

tAtention Deficit Hyperactivity Disorder (ADHD) significantly impairs the performance of students during their early school years. However, the extent of the difference in postural stability between children with ADHD and Typically Developed (TD) children, are still not fully understood. Also, it is clinically important to investigate possible effects of medication on their performances. We recruited 38 children between the ages 6 to 12 with ADHD (15 medically naïve, 23 with current treatment history) and 25 age-matched as TD. Postural stability performance was assessed in four conditions: 1-eyes open, 2-eyes closed, 3-eyes open on foam and 4-eyes closed on foam. A cost-effective mobile force platform was used to evaluate postural performance quantitatively. Results revealed a lower stability performance in the medically-naïve participants especially in trials with eyes open on foam. No significant difference in performance was seen between children with medical treatment history and the TD group. Results suggest that using medication can effectively enhance postural stability for children with ADHD. We found portable balance assessment tools helpful in identifying changes in motor performances among children with ADHD. Our results indicate that further research is needed to understand the exact implications of ADHD on postural stability under different sensory stimuli.

Palmitoylethanolamide as adjunctive therapy for autism: Efficacy and safety results from a randomized controlled trial.

Inflammation as well as glutamate excitotoxicity have been proposed to participate in the propagation of autism. Palmitoylethanolamide (PEA) is an endocannabinoid proven to prevent glutamatergic toxicity and inhibit inflammatory responses simultaneously. The present randomized, parallel group, double-blind placebo-controlled trial is the first study depicted to probe the efficacy of co-treatment with risperidone and PEA over 10 weeks in children with autism. Seventy children (aged 4-12 years) with autism and moderate to severe symptoms of irritability were randomly assigned to two treatment regimens. The study outcomes were measured using the Aberrant Behavior Checklist-Community Edition (ABC-C). At trial endpoint (week 10), combination of PEA and risperidone had superior efficacy in ameliorating the ABC-irritability and hyperactivity/noncompliance symptoms (Cohen's d, 95% confidence interval (CI) = 0.94, 0.41 to 1.46, p = 0.001) compared with a risperidone plus placebo regimen. Interestingly, effect of combination treatment on hyperactivity symptoms was also observed at trial midpoint (week 5) but with a smaller effect size (d = 0.53, p = 0.04) than that at the endpoint (d = 0.94, p = 0.001). Meanwhile, there was a trend toward significance for superior effect of risperidone plus PEA over risperidone plus placebo on inappropriate speech at trial endpoint (d = 0.51, p = 0.051). No significant differences existed between the two treatment groups for the other two ABC-C subscales (lethargy/social withdrawal and stereotypic behavior). The findings suggest that PEA may augment therapeutic effects of risperidone on autism-related irritability and hyperactivity. Future studies are warranted to investigate whether PEA can serve as a stand-alone treatment for autism.

Parent-only Group Cognitive Behavioral Intervention for Children with Anxiety Disorders: A Control Group Study.

OBJECTIVE: Parents play an important role in development and continuation of anxiety disorders in children. Yet the evidence on parent contribution in cognitive behavioral therapy (CBT) for childhood anxiety is limited. This open randomized trial examined the effectiveness of a parent-directed group CBT to manage children with anxiety disorders. METHOD: Parents of 42 children aged 6-12 with primary anxiety disorders were allocated to a six, two-hour weekly intervention and a wait-list (WL) control. The Revised Children's Manifest Anxiety, Children's Depression Inventory, Strengths and Difficulties Questionnaire-Home Version, Depression-Anxiety-Stress Scale, Children Global Assessment Scale, and Global Relational Assessment of Functioning were used to assess children's and parents' functioning and emotional symptoms. Parents completed consumer satisfaction questionnaire. RESULTS: Parents in the CBT group reported significant improvement in their depressive symptoms (p=0.006) and the family functioning (p=0.04), as well as reduction in children's emotional symptoms (p=0.007). Clinician rating of children's functioning showed significant improvement in the CBT group(p=0.001). There was no significant difference in children rating of their anxiety within groups from pre- to post-intervention. Parents were satisfied mostly with the intervention. CONCLUSION: A brief parent-only CBT based intervention can be effective in the management of childhood anxiety.

OBJECTIF: Les parents jouent un rôle important dans le développement et la continuité des troubles anxieux chez les enfants. Et pourtant, les données probantes sur la contribution des parents à la thérapie cognitivo-comportementale (TCC) pour l'anxiété chez les enfants sont limitées. Cet essai randomisé ouvert a examiné l'efficacité d'un groupe de TCC dirigé par des parents pour prendre en charge des enfants souffrant de troubles anxieux. MÉTHODE: Les parents de 42 enfants de 6 à 12 ans souffrant de troubles anxieux primaires ont été affectés à 6 interventions hebdomadaires de 2 heures et à un groupe témoin en liste d'attente (LA). L'échelle révisée de l'anxiété manifeste des enfants, l'inventaire de dépression des enfants, la version maison du questionnaire des forces et difficultés, l'échelle de dépression-anxiété-stress, l'échelle d'évaluation globale des enfants, et l'évaluation relationnelle globale du fonctionnement ont servi à évaluer le fonctionnement et les symptômes émotionnels des enfants et des parents. Les parents ont répondu à un questionnaire sur la satisfaction du consommateur. RÉSULTATS: Les parents du groupe de TCC ont déclaré une amélioration significative de leurs symptômes dépressifs (p = 0,006) et du fonctionnement familial (p = 0,04), ainsi qu'une réduction des symptômes émotionnels des enfants (p = 0,007). L'évaluation du clinicien du fonctionnement des enfants a indiqué une amélioration significative dans le groupe de TCC (p = 0,001). Il n'y avait pas de différence significative de l'évaluation de l'anxiété des enfants au sein des groupes d'avant-après l'intervention. Les parents étaient plutôt satisfaits de l'intervention. CONCLUSION: Une brève intervention réservée aux parents basée sur la TCC peut être efficace pour la prise en charge de l'anxiété chez les enfants.

A Case of Congenital Insensitivity to Pain With Anhidrosis Comorbid With Attention Deficit Hyperactivity Disorder: Clinical Implications for Pathophysiology and Treatment.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disorder caused by a mutation in the neurotrophic tyrosine kinase receptor (NTRK1) gene. CIPA is accompanied by abnormal catecholamine metabolism and decreased blood concentration of dopamine and norepinephrine. Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder of heterogeneous etiology and presentation, and recent reports have suggested a pathophysiological role of neurotrophins in ADHD. Furthermore, dopamine and norepinephrine are known to play major roles in the pathophysiology of ADHD, and the imbalance of monoaminergic and cholinergic systems as an underlying cause of ADHD has recently been studied. Here, we report the case of an 11-year-old boy with CIPA and comorbid ADHD. Our observations have important clinical implications for patients with CIPA. Because of deficiencies in self-control, proper management of these patients necessitates a highly structured and monitored environment, made dually important by possible comorbidity of ADHD.

Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).

Occurrence of chlorinated volatile organic compounds (VOCs) in a sanitary sewer system: Implications for assessing vapor intrusion alternative pathways.

Sewer systems have been recently recognized as potentially important exposure pathways to consider during vapor intrusion assessments; however, this pathway has not been well-characterized and there is need for additional information about the occurrence of volatile organic compounds (VOCs) in sewer systems. This paper reports the results of sewer gas sampling conducted in a sanitary sewer over the years of 2014-2017. Sewer gas samples were collected and analyzed using several different techniques, including TO-15 (grab), TO-17 (passive), Radiello® (passive) and a novel continuous monitoring technique, the Autonomous Rugged Optical Multigas Analyzer (AROMA). The applicability of each of the different approaches used in this study is discussed in the context of investigating sanitary sewers as a vapor intrusion alternative pathway. The data confirmed that trichloroethylene (TCE) concentrations in sewer gas were detected adjacent to and extending hundreds of feet away from a previously defined vapor intrusion area, where TCE was a primary contaminant. TCE concentrations detected in sewer gas ranged from non-detect to 1600µg/m3. Temporal variability was observed in TCE concentrations over timescales that ranged from minutes to months to years at discrete sampling locations. Spatial variability in sewer gas concentrations was also observed throughout the study area. Temporal and spatial variability may be caused by groundwater contamination sources in the study area, as well as sewer gas transport mechanisms.