Basal Gene Expression Levels Research Articles

Differences in inhibitory avoidance, cortisol and brain gene expression in TL and AB zebrafish.

Recently, we established an inhibitory avoidance paradigm in Tupfel Long-Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole-body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post-task. Inhibitory avoidance was assessed in a 3-day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V-shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long-Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance-related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress-axis related genes than TL fish. Tupfel Long-Fin fish showed post-task learning-related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress-related disorders.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Elevated seawater temperatures have a limited impact on the coral immune response following physical damage

Recurrent disturbances on coral reefs that cause injuries, like predation and storm damage, and elevated seawater temperatures reduce coral fitness and immunocompetence. An effective immune response is essential to prevent disease and enhance colony survival. To evaluate how elevated seawater temperatures affect the coral immune response following injury, fragments of Acropora aspera were exposed to ambient (27–29°C) or elevated (32–33.5°C) seawater temperatures for 8 days and subsequently experimentally injured. Expression patterns for 15 immune genes 24 h post-injury revealed that most genes involved in the Toll-like receptor pathway were unaffected by elevated seawater temperatures. Exceptions to this pattern were cFos and cJun, which were upregulated and likely played a role in repair processes, and TRAF-6 and NFκB, which were downregulated suggesting reduced immune function. Components of the complement system were upregulated (millectin, C3) or downregulated (Bf, Tx60, apextrin) in corals at high temperatures. However, corals that also sustained injury, showed normal Tx60 and apextrin expression, suggesting roles in the wounding response. Overall, basal expression levels of immune genes are sufficient to mount a response to injury in the short term, and the immune response of A. aspera following injury is not significantly affected by minor elevations in seawater temperatures.

Expression of drug transporters in human kidney: impact of sex, age, and ethnicity.

BackgroundDifferences in expression of drug transporters in human kidney contribute to changes in pharmacokinetics and toxicokinetics of a variety of drug compounds. The basal expression levels of genes involved in drug transport processes in the kidney introduces differences in bioavailability, distribution, and clearance of drugs, possibly influencing drug efficacy and adverse reactions. Sex differences in gene expression of transporters are a key cause of differences in sex-dependent pharmacokinetics, which may characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Therefore, evaluating the expression of drug transporters in normal human kidneys is important to better understand differences in drug bioavailability, distribution, and clearance of drugs in humans. Other factors such as age and ethnicity may also contribute to individual differences in gene expression of drug transporters in the human kidney.MethodsQuantitative real-time PCR (QRT-PCR) was performed to determine the gene expression of 30 drug transporters in 95 age-matched normal human kidney tissues. Multiple Student’s t-tests (Sidak-Bonferroni correction) and two-way ANOVA (Bonferroni correction) analyses were used to determine statistically significant differences.ResultsIn the 30 transporter genes examined, sex, ethnicity, and age differences in gene expression were exhibited in normal human kidney tissue. These changes in expression were not found to be differentially significant. However, sex-age and sex-ethnicity interactions were found to be statistically significant. For sex-age interactions, SCL22A12 was found to be significantly higher expressed in females <50 years compared to males <50 years. Expression levels of SLC22A2, SLC22A12, SLC6A16, and ABCB6 were significantly higher in females <50 years compared to females ≥50 years. In sex-ethnicity interactions, expression levels of ATP7B and KCNJ8 were found to be significantly higher in African American females compared to European American females. Also, the expression of SLC31A2 was significantly higher in European American males compared to European American females.ConclusionsSex, age, and ethnic differences impacted the expression of drug transporters in normal human kidneys, which suggests that the analysis of gene expression of drug transporters will aid in improving the usage/dosage of drug therapies influencing personalized medicine and susceptibility to adverse drug reactions.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.

Disruption of polyubiquitin gene Ubc leads to attenuated resistance against arsenite-induced toxicity in mouse embryonic fibroblasts

The polyubiquitin gene Ubc is upregulated under oxidative stress induced by arsenite [As(III)]. However, the detailed mechanism of Ubc upregulation and the exact role of ubiquitin (Ub) to protect cells against As(III)-induced toxicity remain unknown. Here, we found that Ubc−/− mouse embryonic fibroblasts (MEFs) exhibited reduced viability under As(III) exposure, although the Nrf2–Keap1 pathway was activated as a cytoprotective response. Intriguingly, due to the reduced polyubiquitination and delayed onset of degradation of Nrf2 in Ubc−/− MEFs, the basal expression levels of Nrf2 target genes were elevated. As(III)-induced accumulation of Ub conjugates occurred in an Nrf2-independent manner, probably due to cellular stress conditions, including reduced proteasomal activity. Increased cellular Ub levels were essential to polyubiquitinate misfolded proteins generated under As(III) exposure and to degrade them by the proteasome. However, when cellular Ub levels decreased, these misfolded proteins were not efficiently polyubiquitinated, but rather accumulated as large protein aggregates inside the cells, causing cytotoxicity. Furthermore, increased activity of the autophagic pathway to clear these aggregates was not observed in Ubc−/− MEFs. Therefore, reduced viability of Ubc−/− MEFs under As(III) exposure may not be due to dysregulation of the Nrf2–Keap1 pathway, but mostly to reduced efficacy to polyubiquitinate and degrade misfolded protein aggregates.

Sexual dimorphism in the expression of mitochondria-related genes in rat heart at different ages.

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Moreover, sex and age are considered major risk factors in the development of CVDs. Mitochondria are vital for normal cardiac function, and regulation of mitochondrial structure and function may impact susceptibility to CVD. To identify potential role of mitochondria in sex-related differences in susceptibility to CVD, we analyzed the basal expression levels of mitochondria-related genes in the hearts of male and female rats. Whole genome expression profiling was performed in the hearts of young (8-week), adult (21-week), and old (78-week) male and female Fischer 344 rats and the expression of 670 unique genes related to various mitochondrial functions was analyzed. A significant (p<0.05) sexual dimorphism in expression levels of 46, 114, and 41 genes was observed in young, adult and old rats, respectively. Gene Ontology analysis revealed the influence of sex on various biological pathways related to cardiac energy metabolism at different ages. The expression of genes involved in fatty acid metabolism was significantly different between the sexes in young and adult rat hearts. Adult male rats also showed higher expression of genes associated with the pyruvate dehydrogenase complex compared to females. In young and adult hearts, sexual dimorphism was not noted in genes encoding oxidative phosphorylation. In old rats, however, a majority of genes involved in oxidative phosphorylation had higher expression in females compared to males. Such basal differences between the sexes in cardiac expression of genes associated with energy metabolism may indicate a likely involvement of mitochondria in susceptibility to CVDs. In addition, female rats showed lower expression levels of apoptotic genes in hearts compared to males at all ages, which may have implications for better preservation of cardiac mass in females than in males.

HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism.

The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9% in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.

MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin.

Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

Betacyanin biosynthetic genes and enzymes are differentially induced by (a)biotic stress in Amaranthus hypochondriacus.

An analysis of key genes and enzymes of the betacyanin biosynthetic pathway in Amaranthus hypochondriacus (Ah) was performed. Complete cDNA sequence of Ah genes coding for cyclo-DOPA 5-O glucosyltransferase (AhcDOPA5-GT), two 4, 5-DOPA-extradiol-dioxygenase isoforms (AhDODA-1 and AhDODA-2, respectively), and a betanidin 5-O-glucosyltransferase (AhB5-GT), plus the partial sequence of an orthologue of the cytochrome P-450 R gene (CYP76AD1) were obtained. With the exception AhDODA-2, which had a closer phylogenetic relationship to DODA-like genes in anthocyanin-synthesizing plants, all genes analyzed closely resembled those reported in related Caryophyllales species. The measurement of basal gene expression levels, in addition to the DOPA oxidase tyrosinase (DOT) activity, in different tissues of three Ah genotypes having contrasting pigmentation levels (green to red-purple) was determined. Additional analyses were performed in Ah plants subjected to salt and drought stress and to two different insect herbivory regimes. Basal pigmentation accumulation in leaves, stems and roots of betacyanic plants correlated with higher expression levels of AhDODA-1 and AhB5-GT, whereas DOT activity levels coincided with pigment accumulation in stems and roots and with the acyanic nature of green plants, respectively, but not with pigmentation in leaves. Although the abiotic stress treatments tested produced changes in pigment levels in different tissues, pigment accumulation was the highest in leaves and stems of drought stressed betacyanic plants, respectively. However, tissue pigment accumulation in stressed Ah plants did not always correlate with betacyanin biosynthetic gene expression levels and/or DOT activity. This effect was tissue- and genotype-dependent, and further suggested that other unexamined factors were influencing pigment content in stressed Ah. The results obtained from the insect herbivory assays, particularly in acyanic plants, also support the proposal that these genes could have functions other than betacyanin biosynthesis.

Unveiling translocation intermediates of RNA polymerase.

Translocation of RNA polymerase (RNAP) is a robust target for regulation of gene expression in prokaryotes and eukaryotes (1⇓–3). During elongation, RNAP frequently encounters a broad range of DNA/RNA conformations (RNA hairpins, curved and cruciform DNA), DNA-binding proteins, DNA lesions, and misincorporation events at the 3′ ends of the RNA. These encounters impede forward translocation, leading to RNAP pausing (3). There are many protein factors that strengthen or weaken pausing by targeting translocation, such as archaeal/eukaryotic Spt5 and bacterial NusG/RfaH (4) or N/Nun proteins of lambdoid phages (3). In metazoa, RNAP II pausing in promoter-proximal regions plays a role in having polymerases in place for a rapid transcription response to environmental stimuli, such as heat-shock or cell differentiation, and maintaining a basal level of gene expression (5, 6). Translocation blocks also initiate RNAP backtracking (7). Backtracking events disengage the 3′ RNA end from the RNAP catalytic site, which stabilizes pausing; this type of event broadly controls gene transcription in eukaryotes (8). Forward translocation of RNAP along DNA has long been regarded as the movement of the RNA–DNA hybrid through the catalytic cleft, which vacates the active center, termed i+1 site, for an NTP binding. In this sense, the mechanism of translocation has been primarily focused on the hybrid movement, with only limited emphasis on the DNA sequences surrounding the hybrid (1, 2, 9). However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7, 10⇓–12). In PNAS, Silva et al. (13) identify an important component of the translocation mechanism using millisecond molecular dynamics (MD) simulation of translocation of yeast RNAP II: Translocation of the hybrid occurs before entry of the template DNA base to the i+1 site, where it can pair …

Intragenic Controls Utilizing Radiation-Induced Alternative Transcript Regions Improves Gene Expression Biodosimetry

Ionizing-radiation exposure can be life threatening if given to the whole body. In addition, whole body radiation exposure can affect large numbers of people such as after a nuclear reactor accident, a nuclear explosion or a radiological terrorist attack. In these cases, an accurate biodosimeter is essential for triage management. One of the problems for biodosimetry in general is the interindividual variation before and after exposure, which can make it challenging to assign an accurate dose. To begin to address this challenge, lymphocyte cell lines were exposed to 0, 1, 2 and 5 Gy ionizing radiation from a ¹³⁷Cs source at a dose rate of 0.6 Gy/min. Alternative transcripts with regions showing large differential responses to ionizing radiation were determined from exon array data. Gene expression analysis was then performed on isolated mRNA using qRT-PCR with normalization to intergenic (PGK1, GAPDH) and novel intragenic regions for candidate radiation-responsive genes, PPM1D and MDM2. Our studies show that the use of a cis-associated expression reference improved the potential dose prediction approximately 2.3-8.3 fold and provided an advantage for dose prediction compared to distantly or trans-located control ionizing radiation nonresponsive genes. This approach also provides an alternative gene expression normalization method to potentially reduce interindividual variations when untreated basal gene expression levels are unavailable. Using associated noninduced regions of ionizing radiation-induced genes provides a way to estimate basal gene expression in the irradiated sample. This strategy can be utilized as a biodosimeter on its own or to enhance other gene expression candidates for biodosimetry. This normalization strategy may also be generally applicable for other quantitative PCR strategies where normalization is required for a particular response.

Microbial Dysbiosis Is Associated with Human Breast Cancer

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.

Estradiol improves cardiovascular function through up-regulation of SOD2 on vascular wall

Epidemiological studies have shown that estrogens have protective effects in cardiovascular diseases, even though the results from human clinical trials remain controversial, while most of the animal experiments confirmed this effect, but the detailed mechanism remains unclear. In this study, we found that estradiol (E2) treatment significantly increases the expression of mitochondrial superoxide dismutase (SOD2) in mice and in vitro in human aorta endothelial cells. Further investigation shows that E2 up-regulates SOD2 through tethering of estrogen receptor (ER) to Sp1 and the increased binding of Sp1 to GC-box on the SOD2 promoter, where ERα responses E2-mediated gene activation, and ERβ maintains basal gene expression level. The E2/ER-mediated SOD2 up-regulation results in minimized ROS generation, which highly favors healthy cardiovascular function. Gene therapy through lentivirus-carried endothelium-specific delivery to the vascular wall in high-fat diet (HFT) mice shows that the SOD2 expression in endothelial cells normalizes E2 deficiency-induced ROS generation with ameliorated mitochondrial dysfunction and vascular damage, while SOD2 knockdown worsens the problem despite the presence of E2, indicating that E2-induced SOD2 expression plays an important vasculoprotective role. To our knowledge, this is the first report for the mechanism by which E2 improves cardiovascular function through up-regulation of SOD2 in endothelial cells. In turn, this suggests a novel gene therapy through lentivirus-carried gene delivery to vascular wall for E2 deficiency-induced cardiovascular damage in postmenopausal women.

Effects of voluntary alcohol drinking on corticotropin-releasing factor and preprodynorphin mRNA levels in the central amygdala of Sardinian alcohol-preferring rats

The stress-response corticotropin-releasing factor (CRF) and dynorphin systems are critically involved in alcohol drinking and “anxiety”-related behaviors. Selectively bred Sardinian alcohol-preferring (sP) rats display high inherent “anxiety”-related behaviors, in comparison with their alcohol-nonpreferring counterpart (sNP rats). The present study was undertaken to investigate: (1) if there were genetically determined differences in basal gene expression levels of CRF, CRF-R1, preprodynorphin (ppDyn) and kappa opioid receptor (KOP-r) between sP and sNP rats; specifically, mRNA levels of the above genes were measured in the central amygdala (CeA), hypothalamus and other stress responsive and mesolimbic regions of alcohol-naive sP and sNP rats; and (2) if the above mRNA levels were altered by voluntary alcohol drinking in sP rats exposed to the standard, homecage 2-bottle “alcohol vs. water” choice regimen 24h/day for 17 days. Higher basal CRF mRNA levels were found only in CeA of alcohol-naive sP rats, compared with sNP rats; these levels were decreased after alcohol consumption. In contrast, ppDyn mRNA levels in CeA of sP rats were increased by alcohol consumption, but with no basal difference from sNP rats. Although higher basal ppDyn mRNA levels were found in hypothalamus of sP rats, compared with sNP rats, there was no alteration after alcohol drinking in sP rats. No difference for the above mRNA levels was observed in other regions, including nucleus accumbens shell or core, caudate-putamen, ventral tegmental area and medial/basolateral amygdala, between the two rat lines before or after alcohol consumption. Our results demonstrate the existence of genetically determined high basal CRF mRNA levels in CeA of sP rats. Alcohol consumption decreased CeA CRF mRNA levels with parallel increases in CeA ppDyn mRNA levels.

Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor

The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time- and money-saving tools that aid the efficient selection of cellular specimens--in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell-based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage-like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor-β and elevated levels of Sox9.

Determination of MDR1 gene expression for prediction of chemotherapy tolerance and treatment outcome in dogs with lymphoma

The multidrug resistance gene 1(MDR1) expression levels were analysed in 27 dogs with different types of malignant lymphomas receiving a standard chemotherapy protocol. Blood samples were used for MDR1 real-time PCR expression analysis. Treatment tolerance and outcome were evaluated on a regular basis by clinical examination and client questioning. Dogs developing severe adverse effects under treatment showed significantly lower basal MDR1 gene expression levels when compared with those who tolerated the drugs well. In the longitudinal MDR1 gene expression analysis during treatment, four dogs showed a greater than two-fold MDR1 up-regulation, compared to baseline expression. All four of these dogs, but none of the others, showed disease progression. In conclusion, basal and follow-up MDR1 gene expression levels could be of predictive value for the occurrence of severe adverse drug reactions and/or the development of MDR during chemotherapy for lymphoma in dogs.

THU0054 Role of Fra-2 in TLR8-Mediated TIMP-1 Production in Systemic Sclerosis

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells. We and others have shown that monocytes along with fibroblasts...

Abstract 4093: Growth factors regulated by the aryl hydrocarbon receptor in highly metastatic head and neck squamous cell carcinoma cell lines.

Abstract The aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, is implicated in mediating pleotropic cellular effects in response to a variety of AHR ligands. Recent evidence has implicated the AHR in tumor initiation, promotion and progression as well. Enhanced AHR expression has been associated with dysregulated cellular proliferation, enhanced cell motility and loss of cell-cell contact. Previous studies from this laboratory have revealed considerable constitutive and ligand-inducible AHR transcriptional activity in head and neck squamous cell carcinoma cell lines (HNSCC). Subsequently, antagonism of AHR activity was found to greatly attenuate the highly metastatic and proliferative phenotype of these cells, thereby suggesting that the AHR plays a pivotal role in dictating the ‘aggressive’ phenotype of these cells. Hence, this study sought to use a whole genome expression profiling based approach to identify putative novel targets of the AHR associated with cancer cell survival and tumor invasiveness. Given previous documented evidence, from other studies in the literature, of the involvement of growth factors in dictating the aggressive phenotype of various cancers, this study sought to focus specifically on the growth factor targets regulated by AHR. Additionally, enhanced growth factor expression, along with high inflammatory cytokine expression, has been found to play a prominent role in dictating tumor cell malignancy. Given previous evidence from our laboratory of high inflammatory cytokine expression (IL1B, IL6) in HNSCC cell lines, the expression of growth factors was also monitored in the presence of an inflammatory signal (IL1B) and an AHR agonist (TCDD). Three growth factor targets were considered; amphiregulin (AREG), epiregulin (EREG) and platelet-derived growth factor A (PDGFA) based on expression profiling and from previous studies. Quantitative PCR analysis revealed an attenuation of basal and/or induced gene expression levels of these growth factors following AHR antagonism, either by AHR antagonist treatment or by siRNA mediated AHR knock down. ELISA analysis revealed an attenuation of both basal and induced protein levels of the growth factors examined in these cell lines. In silico analysis revealed these growth factors to possess dioxin-like response elements in regions upstream of their promoter. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene also exhibited induction of gene expression of the AHR growth factors examined. TCDD+IL1B in combination caused a significant increase in the expression of all growth factors examined with CH223191 causing an attenuation of their expression. In conclusion, AREG, EREG and PDGFA were identified as three key growth factor targets of the AHR associated with metastatic phenotype of head and neck cancers. Citation Format: Kaarthik John, Jarod Hughes, Tejas Lahoti, Wagner Kelly, Perdew Gary. Growth factors regulated by the aryl hydrocarbon receptor in highly metastatic head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2013-4093