Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks.

BACKGROUND: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. METHOD: Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. RESULTS: All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of cytokines IL6 and CXCL8 (IL8) was not different between the lines regardless of CFTR genotype. All lines responded to TNFα and IL1ß by increasing IL6 and CXCL8 mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFα, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR's absence, others required stimulation for differences to be apparent. CONCLUSION: Together, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.

Small adipose stores in cystic fibrosis mice are characterized by reduced cell volume, not cell number.

Cystic fibrosis (CF) is a lethal genetic disorder that affects many organ systems of the body, including various endocrine and exocrine tissues. Health and survival positively associate with body mass, and as a consequence, CF clinical care includes high-fat, high-calorie diets to maintain and increase adipose tissue stores. Such strategies have been implemented without a clear understanding of the cause and effect relationship between body mass and patients' health. Here, we used CF mouse models, which display small adipose stores, to begin examining body fat as a prelude into mechanistic studies of low body growth in CF, so that optimal therapeutic strategies could be developed. We reasoned that low adiposity must result from reduced number and/or volume of adipocytes. To determine relative contribution of either mechanism, we quantified volume of intraperitoneal and subcutaneous adipocytes. We found smaller, but not fewer, adipocytes in CF compared with wild-type (WT) animals. Specifically, intraperitoneal CF adipocytes were one-half the volume of WT cells, whereas subcutaneous cells were less affected by the Cftr genotype. No differences were found in cell types between CF and WT adipose tissues. Adipose tissue CFTR mRNA was detected, and we found greater CFTR expression in intraperitoneal depots as compared with subcutaneous samples. RNA sequencing revealed that CF adipose tissue exhibited lower expression of several key genes of adipocyte function ( Lep, Pck1, Fas, Jun), consistent with low triglyceride storage. The data indicate that CF adipocytes contain fewer triglycerides than WT cells, and a role for CFTR in these cells is proposed. NEW & NOTEWORTHY Adipocytes in cystic fibrosis mice exhibit smaller size due to low triglyceride storage. Adipocyte cell number per fat pad is similar, implying triglyceride storage problem. The absence of CFTR function in adipose tissue has been proposed as a direct link to low triglyceride storage in cystic fibrosis.

Chronic hindlimb suspension unloading markedly decreases turnover rates of skeletal and cardiac muscle proteins and adipose tissue triglycerides.

We previously showed that a single bolus of "doubly-labeled" water ((2)H2 (18)O) can be used to simultaneously determine energy expenditure and turnover rates (synthesis and degradation) of tissue-specific lipids and proteins by modeling labeling patterns of protein-bound alanine and triglyceride-bound glycerol (Bederman IR, Dufner DA, Alexander JC, Previs SF. Am J Physiol Endocrinol Metab 290: E1048-E1056, 2006). Using this novel method, we quantified changes in the whole body and tissue-specific energy balance in a rat model of simulated "microgravity" induced by hindlimb suspension unloading (HSU). After chronic HSU (3 wk), rats exhibited marked atrophy of skeletal and cardiac muscles and significant decrease in adipose tissue mass. For example, soleus muscle mass progressively decreased 11, 43, and 52%. We found similar energy expenditure between control (90 ± 3 kcal · kg(-1)· day(-1)) and hindlimb suspended (81 ± 6 kcal/kg day) animals. By comparing food intake (∼ 112 kcal · kg(-1) · day(-1)) and expenditure, we found that animals maintained positive calorie balance proportional to their body weight. From multicompartmental fitting of (2)H-labeling patterns, we found significantly (P < 0.005) decreased rates of synthesis (percent decrease from control: cardiac, 25.5%; soleus, 70.3%; extensor digitorum longus, 44.9%; gastrocnemius, 52.5%; and adipose tissue, 39.5%) and rates of degradation (muscles: cardiac, 9.7%; soleus, 52.0%; extensor digitorum longus, 27.8%; gastrocnemius, 37.4%; and adipose tissue, 50.2%). Overall, HSU affected growth of young rats by decreasing the turnover rates of proteins in skeletal and cardiac muscles and adipose tissue triglycerides. Specifically, we found that synthesis rates of skeletal and cardiac muscle proteins were affected to a much greater degree compared with the decrease in degradation rates, resulting in large negative balance and significant tissue loss. In contrast, we found a small decrease in adipose tissue triglyceride synthesis paired with a large decrease in degradation, resulting in smaller negative energy balance and loss of fat mass. We conclude that HSU in rats differentially affects turnover of muscle proteins vs. adipose tissue triglycerides.

Psychometric properties of the Generalized Anxiety Disorder Inventory in a Canadian sample.

The Generalized Anxiety Disorder Inventory is a recently developed self-report measure that assesses symptoms of generalized anxiety disorder. Its psychometric properties have not been investigated further since its original development. The current study investigated its psychometric properties in a Canadian student/community sample. Exploratory principal component analysis replicated the original three-component structure. The total scale and subscales demonstrated excellent internal consistency reliability (α = 0.84-0.94) and correlated strongly with the Penn State Worry Questionnaire (r = 0.41-0.74, all ps <0.001) and Generalized Anxiety Disorder-7 (r = 0.55-0.84, all ps <0.001). However, only the total scale and cognitive subscale (r = 0.48-0.49, all ps <0.05) significantly predicted generalized anxiety disorder diagnosis established by diagnostic interview. The somatic subscale in particular may require revision to improve predictive validity. Revision may also be necessary given changes in required somatic symptoms for generalized anxiety disorder diagnostic criteria in more recent versions of the Diagnostic and Statistical Manual of Mental Disorders (i.e. although major changes occurred from Diagnostic and Statistical Manual of Mental Disorders-III-R to Diagnostic and Statistical Manual of Mental Disorders-IV, changes in Diagnostic and Statistical Manual of Mental Disorders-5 were minimal) and the possibility of changes in the upcoming 11th revision of the International Classification of Diseases.

ADEMA: an algorithm to determine expected metabolite level alterations using mutual information.

Metabolomics is a relatively new "omics" platform, which analyzes a discrete set of metabolites detected in bio-fluids or tissue samples of organisms. It has been used in a diverse array of studies to detect biomarkers and to determine activity rates for pathways based on changes due to disease or drugs. Recent improvements in analytical methodology and large sample throughput allow for creation of large datasets of metabolites that reflect changes in metabolic dynamics due to disease or a perturbation in the metabolic network. However, current methods of comprehensive analyses of large metabolic datasets (metabolomics) are limited, unlike other "omics" approaches where complex techniques for analyzing coexpression/coregulation of multiple variables are applied. This paper discusses the shortcomings of current metabolomics data analysis techniques, and proposes a new multivariate technique (ADEMA) based on mutual information to identify expected metabolite level changes with respect to a specific condition. We show that ADEMA better predicts De Novo Lipogenesis pathway metabolite level changes in samples with Cystic Fibrosis (CF) than prediction based on the significance of individual metabolite level changes. We also applied ADEMA's classification scheme on three different cohorts of CF and wildtype mice. ADEMA was able to predict whether an unknown mouse has a CF or a wildtype genotype with 1.0, 0.84, and 0.9 accuracy for each respective dataset. ADEMA results had up to 31% higher accuracy as compared to other classification algorithms. In conclusion, ADEMA advances the state-of-the-art in metabolomics analysis, by providing accurate and interpretable classification results.

Time course of hepatic gluconeogenesis during hindlimb suspension unloading.

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.

A new metabolomics analysis technique: steady-state metabolic network dynamics analysis.

With the recent advances in experimental technologies, such as gas chromatography and mass spectrometry, the number of metabolites that can be measured in biofluids of individuals has markedly increased. Given a set of such measurements, a very common task encountered by biologists is to identify the metabolic mechanisms that lead to changes in the concentrations of given metabolites and interpret the metabolic consequences of the observed changes in terms of physiological problems, nutritional deficiencies, or diseases. In this paper, we present the steady-state metabolic network dynamics analysis (SMDA) approach in detail, together with its application in a cystic fibrosis study. We also present a computational performance evaluation of the SMDA tool against a mammalian metabolic network database. The query output space of the SMDA tool is exponentially large in the number of reactions of the network. However, (i) larger numbers of observations exponentially reduce the output size, and (ii) exploratory search and browsing of the query output space is provided to allow users to search for what they are looking for.

Altered de novo lipogenesis contributes to low adipose stores in cystic fibrosis mice.

Cystic fibrosis (CF) mouse models exhibit exocrine pancreatic function, yet they do not develop adipose stores to the levels of non-CF mice. CF mice homozygous for the Cftr mutation (F508del) at 3 wk (postweaning) and 6 wk (young adult) of age had markedly less adipose tissue than non-CF mice. Food intake was markedly lower in 3-wk-old CF mice but normalized by 6 wk of age. Both 3- and 6-wk-old mice had dietary lipid absorption and fecal lipid excretion comparable to non-CF mice. Hepatic de novo lipogenesis (DNL), determined by (2)H incorporation, was reduced in CF mice. At 3 wk, F508del mice had significantly decreased DNL of palmitate and stearate, by 83% and 80%, respectively. By 6 wk, DNL rates in non-CF mice remained unchanged compared with 3-wk-old mice, while DNL rates of F508del mice were still reduced, by 33% and 40%, respectively. Adipose tissue fatty acid (FA) profiles were comparable in CF and non-CF mice, indicating that adipose differences are quantitative, not qualitative. A correspondingly lower content of (2)H-labeled FA was found in CF adipose tissue, consistent with reduced deposition of newly made hepatic triglycerides and/or decreased adipose tissue lipogenesis. Hepatic transcriptome analysis revealed lower mRNA expression from several genes involved in FA biosynthesis, suggesting downregulation of this pathway as a mechanism for the reduced lipogenesis. These novel data provide a model for altered lipid metabolism in CF, independent of malabsorption, and may partly explain the inability of pancreatic enzyme replacement therapy to completely restore normal body mass to CF patients.

A review of the genotoxicity of ethylbenzene.

Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (IARC class 2B). It induces tumours in rats and mice, but neither the relevance of these tumours to humans nor their mechanism of induction is clear. Considering the carcinogenic potential of ethylbenzene, it is of interest to determine whether there is sufficient data to characterize its mode of action as either genotoxic or non-genotoxic. A review of the currently available genotoxicity data is assessed. Ethylbenzene is not a bacterial mutagen, does not induce gene conversion or mutations in yeast and does not induce sister chromatid exchanges in CHO cells. Ethylbenzene is not clastogenic in CHO or rat liver cell lines but was reported to induce micronuclei in SHE cells in vitro. No evidence for genotoxicity has been seen in humans exposed to relatively high levels of ethylbenzene. Mouse lymphoma gene mutation studies produced a mixed series of responses that have proved difficult to interpret. An increase in morphological transformation of SHE cells was also found. Results from a more relevant series of in vivo genotoxicity studies, including acute and sub-chronic micronucleus tests and the mouse liver UDS assay, indicate a lack of in vivo genotoxic activity. The composite set of results from both in vitro and in vivo tests known to assess direct damage to DNA have been predominantly negative in the absence of excessive toxicity. The available data from the standard battery of genotoxicity assays do not support a genotoxic mechanism for ethylbenzene-induced kidney, liver or lung tumors in rats and mice.

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity.

The performance of a battery of three of the most commonly used in vitro genotoxicity tests--Ames+mouse lymphoma assay (MLA)+in vitro micronucleus (MN) or chromosomal aberrations (CA) test--has been evaluated for its ability to discriminate rodent carcinogens and non-carcinogens, from a large database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. We re-evaluated many (113 MLA and 30 CA) previously published genotoxicity results in order to categorise the performance of these assays using the response categories we established. The sensitivity of the three-test battery was high. Of the 553 carcinogens for which there were valid genotoxicity data, 93% of the rodent carcinogens evaluated in at least one assay gave positive results in at least one of the three tests. Combinations of two and three test systems had greater sensitivity than individual tests resulting in sensitivities of around 90% or more, depending on test combination. Only 19 carcinogens (out of 206 tested in all three tests, considering CA and MN as alternatives) gave consistently negative results in a full three-test battery. Most were either carcinogenic via a non-genotoxic mechanism (liver enzyme inducers, peroxisome proliferators, hormonal carcinogens) considered not necessarily relevant for humans, or were extremely weak (presumed) genotoxic carcinogens (e.g. N-nitrosodiphenylamine). Two carcinogens (5-chloro-o-toluidine, 1,1,2,2-tetrachloroethane) may have a genotoxic element to their carcinogenicity and may have been expected to produce positive results somewhere in the battery. We identified 183 chemicals that were non-carcinogenic after testing in both male and female rats and mice. There were genotoxicity data on 177 of these. The specificity of the Ames test was reasonable (73.9%), but all mammalian cell tests had very low specificity (i.e. below 45%), and this declined to extremely low levels in combinations of two and three test systems. When all three tests were performed, 75-95% of non-carcinogens gave positive (i.e. false positive) results in at least one test in the battery. The extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity may be induced (whether it is relevant for the whole animal or human) and using weight of evidence approaches to assess the carcinogenic risk from a positive genotoxicity signal. It also highlights deficiencies in the current prediction from and understanding of such in vitro results for the in vivo situation. It may even signal the need for either a reassessment of the conditions and criteria for positive results (cytotoxicity, solubility, etc.) or the development and use of a completely new set of in vitro tests (e.g. mutation in transgenic cell lines, systems with inherent metabolic activity avoiding the use of S9, measurement of genetic changes in more cancer-relevant genes or hotspots of genes, etc.). It was very difficult to assess the performance of the in vitro MN test, particularly in combination with other assays, because the published database for this assay is relatively small at this time. The specificity values for the in vitro MN assay may improve if data from a larger proportion of the known non-carcinogens becomes available, and a larger published database of results with the MN assay is urgently needed if this test is to be appreciated for regulatory use. However, specificity levels of <50% will still be unacceptable. Despite these issues, by adopting a relative predictivity (RP) measure (ratio of real:false results), it was possible to establish that positive results in all three tests indicate the chemical is greater than three times more likely to be a rodent carcinogen than a non-carcinogen. Likewise, negative results in all three tests indicate the chemical is greater than two times more likely to be a rodent non-carcinogen than a carcinogen. This RP measure is considered a useful tool for industry to assess the likelihood of a chemical possessing carcinogenic potential from batteries of positive or negative results.

Review of the genotoxicity of styrene in humans.

Styrene (CAS No. 100-42-5) is an important industrial chemical for which positive results have been reported in in vitro and in vivo genotoxicity assays. Styrene-exposed workers have been studied extensively over two decades for the induction of various types of genotoxic effects. The outcomes of these studies have been conflicting, and where positive responses have been reported, it has proved difficult to demonstrate clear relationships between levels of damage reported and exposure levels. In this review, we have assessed studies addressing mutagenicity (chromosome aberrations, micronuclei and gene mutations) and other endpoints (sister chromatid exchanges, DNA breaks and DNA adducts) using criteria derived from the IPCS guidelines for the conduct of human biomonitoring studies. Based on the re-evaluated outcomes, the data are not convincing that styrene induces gene mutations. The evidence for induction of clastogenicity in occupationally exposed workers is less clear, with a predominant lack of induction of micronuclei in different studies, but conflicting responses in chromosome aberration assays. The results of numerous studies on sister chromatid exchanges do not provide evidence of a clear positive response, despite these being induced in animals exposed to styrene at high concentrations. However, there is evidence that both DNA adducts and DNA single strand breaks are induced in styrene workers. These types of damage are considered indicative of exposure of the target cells and interaction with cellular DNA but do not necessarily result in heritable changes. There is evidence that the metabolism of styrene in humans is affected by genetic polymorphisms of metabolizing genes and that these polymorphisms affect the outcome of in vitro mutagenicity studies on styrene. Therefore, studies that have addressed the potential of this factor to affect in vivo responses were considered. To date, there are no consistent relationships between genetic polymorphisms and induction of genotoxicity by styrene in humans, but further work is warranted on larger samples. The analyses of individual studies, together with a consideration of dose-response relationships and the lack of a common profile of positive responses for the various endpoints in different studies, provide no clear evidence that styrene exposure in workers results in detectable levels of mutagenic damage. However, evidence of exposure to genotoxic metabolites is demonstrated by the formation of DNA adducts and strand breaks.

Review of the in vivo genotoxicity tests performed with styrene.

Results from new genotoxicity tests in laboratory animals have necessitated a comprehensive re-evaluation of the mutagenic potential of styrene in vivo. Available data suggest that styrene, after being metabolized to styrene oxide, is weakly positive in indicator tests detecting DNA adducts, DNA strand breaks and sister chromatid exchanges (SCEs). There is no convincing evidence of styrene clastogenicity in experimental animals when the quality of the studies and the plausibility of the test results are considered. Equivocal results were obtained after exposure to high doses causing lethality. A recently published in vivo micronucleus test (MNT) in bone marrow cells of mice conforming to the current OECD guideline was clearly negative. Consequently, our evaluation of the published genotoxicity data comes to the conclusion that styrene at high doses can induce genotoxic effects in indicator tests. These DNA effects depend upon the exposure levels of the target cells, the metabolic activation to styrene oxide and the efficiency of detoxification. Mutagenic effects of styrene can only be expected under extreme exposure conditions if styrene oxide is not efficiently detoxified and primary DNA lesions are not completely repaired. However, there is no clear evidence that styrene induces mutagenic/clastogenic effects in vivo when tested under appropriate test conditions.

Readiness for cessation of drug use among recent attenders and nonattenders of a needle exchange program.

Needle exchange programs (NEPs) represent a bridge to drug abuse treatment. NEP attenders tend to have more severe drug problems, however, and may be less ready to reduce their drug use than other drug users. This study investigated the relationship between NEP attendance and readiness for cessation of drug use. Since the period from 1988 through 1989, a community-based sample of injection drug users (IDUs) in Baltimore has undergone semiannual interview-administered questionnaires and HIV testing. A total of 288 IDUs completed a questionnaire on readiness for cessation of drug use. Readiness for drug use cessation was assessed from a 28-item validated scale of problem drug use and intention to quit, based on the "stages of change" model. Logistic regression was used to determine factors associated with readiness for cessation of drug use, including sociodemographics, drug use behaviors, and NEP attendance. Thirty percent of respondents attended the NEP in the past month. Stage of change in readiness for cessation of drug use did not differ between NEP attenders and nonattenders (OR = 0.9; 95% CI: 0.5-1.6). Similar proportions of persons recently attending and not attending the NEP were classified as ready to stop drug use (about 30%). In multivariate analysis, readiness for cessation of drug use was associated with speedball injection and previous enrollment in drug treatment but not with NEP attendance. NEP attenders, although exhibiting characteristics consistent with more severe drug dependence, were as motivated for cessation of drug use as were nonattenders. These findings suggest that formal collaboration between NEPs and drug treatment programs could increase the proportion of IDUs in treatment.