Nicotine exposure caused significant transgenerational heritable behavioral changes in Caenorhabditis elegans

Passive and active exposure to tobacco smoking among youth is directly associated with immediate as well as long-term health deterioration. Despite all public health policies and efforts, the percentage of teenage smokers is still relatively high, especially in developing countries. Very few, if any, studies have been done on the transgenerational effect of nicotine exposed during the more sensitive, early developmental stages. We employed C. elegans as a biological model to study the multigenerational impact of chronic nicotine exposure. Nicotine treatment was limited to N2 hermaphrodites of the F0 generation. Exposure was limited to the larval period L1-L4 (~31 hours) after which worms were transferred to a fresh NGM plate. N2 hermaphrodites at L4 developmental stage were used for behavioral analysis across three generations: F0, F1, and F2. Our results show that nicotine was associated with changes in sinusoidal locomotion, speed, and body bends in L4 larvae in all three tested generations. These behavioral alterations were not restricted to F0, but were observed in F1 and F2 generations which were never exposed to nicotine. Our study is the first to reveal that nicotine addiction is heritable using C. elegans as a model organism. These results underscored the sensitivity of early development stages, with hope to spread more awareness to encourage the avoidance of nicotine exposure, especially at a young age

Nicotine exposure caused significant transgenerational heritable behavioral changes in Caenorhabditis elegans

Passive and active exposure to tobacco smoking among youth is directly associated with immediate as well as long-term health deterioration. Despite all public health policies and efforts, the percentage of teenage smokers is still relatively high, especially in developing countries. Very few, if any, studies have been done on the transgenerational effect of nicotine exposed during the more sensitive, early developmental stages. We employed C. elegans as a biological model to study the multigenerational impact of chronic nicotine exposure. Nicotine treatment was limited to N2 hermaphrodites of the F0 generation. Exposure was limited to the larval period L1-L4 (~31 hours) after which worms were transferred to a fresh NGM plate. N2 hermaphrodites at L4 developmental stage were used for behavioral analysis across three generations: F0, F1, and F2. Our results show that nicotine was associated with changes in sinusoidal locomotion, speed, and body bends in L4 larvae in all three tested generations. These behavioral alterations were not restricted to F0, but were observed in F1 and F2 generations which were never exposed to nicotine. Our study is the first to reveal that nicotine addiction is heritable using C. elegans as a model organism. These results underscored the sensitivity of early development stages, with hope to spread more awareness to encourage the avoidance of nicotine exposure, especially at a young age

Dose-Dependent Effects of GLD-2 and GLD-1 on Germline Differentiation and Dedifferentiation in the Absence of PUF-8

PUMILIO/FBF (PUF) proteins have a conserved function in stem cell regulation. Caenorhabditis elegans PUF-8 protein inhibits the translation of target mRNAs by interacting with PUF binding element (PBE) in the 30 untranslated region (30 UTR). In this work, an in silico analysis has identified gld-2 [a poly(A) polymerase] as a putative PUF-8 target. Biochemical and reporter analyses showed that PUF-8 specifically binds to a PBE in gld-2 3 0 UTR and represses a GFP reporter gene carrying gld-2 3 0 UTR in the C. elegans mitotic germ cells. GLD-2 enhances meiotic entry at least in part by activating GLD-1 (a KH motif-containing RNA-binding protein). Our genetic analyses also demonstrated that heterozygous gld-2(+/−) gld-1(+/−) genes in the absence of PUF-8 are competent for meiotic entry (early differentiation), but haplo-insufficient for the meiotic division (terminal differentiation) of spermatocytes. Indeed, the arrested spermatocytes return to mitotic cells via dedifferentiation, which results in germline tumors. Since these regulators are broadly conserved, we thus suggest that similar molecular mechanisms may control differentiation, dedifferentiation, and tumorigenesis in other organisms, including humans

A Comprehensive Approach to Identify Reliable Reference Gene Candidates to Investigate the Link between Alcoholism and Endocrinology in Sprague-Dawley Rats

Gender and hormonal differences are often correlated with alcohol dependence and related complications like addiction and breast cancer. Estrogen (E2) is an important sex hormone because it serves as a key protein involved in organism level signaling pathways. Alcoholism has been reported to affect estrogen receptor signaling; however, identifying the players involved in such multi-faceted syndrome is complex and requires an interdisciplinary approach. In many situations, preliminary investigations included a straight forward, yet informative biotechniques such as gene expression analyses using quantitative real time PCR (qRT-PCR). The validity of qRT-PCR-based conclusions is affected by the choice of reliable internal controls. With this in mind, we compiled a list of 15 commonly used housekeeping genes (HKGs) as potential reference gene candidates in rat biological models. A comprehensive comparison among 5 statistical approaches (geNorm, dCt method, NormFinder, BestKeeper, and RefFinder) was performed to identify the minimal number as well the most stable reference genes required for reliable normalization in experimental rat groups that comprised sham operated (SO), ovariectomized rats in the absence (OVX) or presence of E2 (OVXE2). These rat groups were subdivided into subgroups that received alcohol in liquid diet or isocalroic control liquid diet for 12 weeks. Our results showed that U87, 5S rRNA, GAPDH, and U5a were the most reliable gene candidates for reference genes in heart and brain tissue. However, different gene stability ranking was specific for each tissue input combination. The present preliminary findings highlight the variability in reference gene rankings across different experimental conditions and analytic methods and constitute a fundamental step for gene expression assays

Descriptive statistics of Ct values for heart and brain samples.

<p>(A) 48 samples divided into 12 groups for heart and brain tissue combined. (B) 24 samples for 6 groups for heart tissue. (C) 24 samples for 6 groups for brain tissue. Mean Ct values calculated from raw qRT-PCR output for the 15 candidate genes in 6 experimental groups of SD rats (as described in methods). 50% of the values are included in the box. The median is represented by the line in the box. The interquartile range is bordered by the upper and lower edges, which indicate the 75th and 25th percentiles, respectively. The whiskers are inclusive of the maximal and minimal values, but exclusive of the outliers, represented as circles.</p

Ranking of 10 reference gene candidates based on BestKeeper.

<p>Two criteria are considered: Pearson’s correlation coefficient and BestKeeper computed SD values. The stability of a gene is directly proportional to the [r] value, while it is inversely proportional to the SD value.</p><p>Note: Total sample number (n), Geometric Mean (GM), AM (Arithmetic Mean), Standard Deviation (SD), Coefficient of Varience % (CV), Pearson’s correlation coefficient [r], P<0.05.</p

Quantitative and qualitative analysis based on geNorm.

<p>(A) Ranking of the 15 gene candidates based on the M-value. Three inputs were used for analysis: Heart and brain combined (48 samples/12 groups), heart alone (24 samples/6 groups), and brain alone (24 samples/6 groups). (B) Determination of the minimal number of reference genes based on V-value for the 3 input combinations. Y-axis represents the ratio of (V_n/V_n+1) where 0.15 is the cutoff value. X-axis: V_i/j where “i” starts with 2 genes and “j” starts with 3. geNorm starts by a gene pair, and tests whether the inclusion of a 3^rd gene adds significant variation. This process is repeated to cover all the genes in the list.</p

Summary of mean and SD values of gene pairwise comparison using the dCt method for 15 reference gene candidates.

<p>Summary of mean and SD values of gene pairwise comparison using the dCt method for 15 reference gene candidates.</p

The standard deviations (SD) for each of the 15 gene candidates in descending order.

<p>The values for each input case are shown separately.</p

A summary of the 15 HKG (housekeeping genes) considered as reference gene candidates in SD rats.

<p>A summary of the 15 HKG (housekeeping genes) considered as reference gene candidates in SD rats.</p