Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells.

The purinergic system is fundamental in the tumor microenvironment, since it regulates tumor cell interactions with the immune system, as well as growth and differentiation in autocrine-paracrine responses. Here, we investigated the role of the adenosine A2B receptor (A2BR) in ovarian carcinoma-derived cells' (OCDC) properties. From public databases, we documented that high A2BR expression is associated with a better prognostic outcome in ovarian cancer patients. In vitro experiments were performed on SKOV-3 cell line to understand how A2BR regulates the carcinoma cell phenotype associated with cell migration. RT-PCR and Western blotting revealed that the ADORA2B transcript (coding for A2BR) and A2BR were expressed in SKOV-3 cells. Stimulation with BAY-606583, an A2BR agonist, induced ERK1/2 phosphorylation, which was abolished by the antagonist PSB-603. Pharmacological activation of A2BR reduced cell migration and actin stress fibers; in agreement, A2BR knockdown increased migration and enhanced actin stress fiber expression. Furthermore, the expression of E-cadherin, an epithelial marker, increased in BAY-606583-treated cells. Finally, cDNA microarrays revealed the pathways mediating the effects of A2BR activation on SKOV-3 cells. Our results showed that A2BR contributed to maintaining an epithelial-like phenotype in OCDC and highlighted this purinergic receptor as a potential biomarker.

Functional expression of P2Y2 receptors in mouse ovarian surface epithelium (OSE).

Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology.

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice.

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.

Mouse corticospinal system comprises different functional neuronal ensembles depending on their hodology.

BACKGROUND: Movement performance depends on the synaptic interactions generated by coherent parallel sensorimotor cortical outputs to different downstream targets. The major outputs of the neocortex to subcortical structures are driven by pyramidal tract neurons (PTNs) located in layer 5B. One of the main targets of PTNs is the spinal cord through the corticospinal (CS) system, which is formed by a complex collection of distinct CS circuits. However, little is known about intracortical synaptic interactions that originate CS commands and how different populations of CS neurons are functionally organized. To further understand the functional organization of the CS system, we analyzed the activity of unambiguously identified CS neurons projecting to different zones of the same spinal cord segment using two-photon calcium imaging and retrograde neuronal tracers. RESULTS: Sensorimotor cortex slices obtained from transgenic mice expressing GCaMP6 funder the Thy1 promoter were used to analyze the spontaneous calcium transients in layer 5 pyramidal neurons. Distinct subgroups of CS neurons projecting to dorsal horn and ventral areas of the same segment show more synchronous activity between them than with other subgroups. CONCLUSIONS: The results indicate that CS neurons projecting to different spinal cord zones segregated into functional ensembles depending on their hodology, suggesting that a modular organization of CS outputs controls sensorimotor behaviors in a coordinated manner.

Experimental polycystic ovarian syndrome is associated with reduced expression and function of P2Y2 receptors in rat theca cells.

Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 µM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS.

Steroid Receptors and Aromatase Gene Expression in Different Brain Areas of Copulating and Sexually Sluggish Male Rats.

INTRODUCTION: Sexually sluggish (SS) males have been identified in several species of mammals including rats. These animals take more than 30 minutes to ejaculate; they do not ejaculate or do so inconsistently despite being tested repeatedly with sexually receptive females. Different brain areas and hormones play an important role in the control of male sexual behavior. AIMS: Determine gene expression for the androgen receptor (AR), the estrogen receptor alpha (ERα), the progesterone receptor (PR), and the aromatase enzyme (ARO), in brain regions important in the control of male sexual behavior including the medial preoptic area (MPOA), the amygdala (AMG), the olfactory bulb (OB), and, as a control, the cortex (CTX) of copulating (C) and SS male rats. METHODS: Males that ejaculated within 30 minutes in three tests with receptive females were included in the C group, while those males that ejaculated in one or none of the four tests were included in the SS group. RNA was isolated 1 week after the last test of sexual behavior, and cDNA was synthesized from the brain areas listed above. MAIN OUTCOMES MEASURES: Expression of the AR, ERα, PR, and ARO genes was determined by quantitative polymerase chain reaction (qPCR). Cyclophilin A (CycA) and tyrosine 3-monooxygenase-tryptophan activation protein zeta (Ywhaz) were housekeeping genes used to determine relative gene expression with the 2(-ΔΔCt) method. RESULTS: The expression of mRNA for AR and ARO increased in the MPOA of SS males. ARO mRNA was increased in the AMG of SS males. In the OB, ERα mRNA was increased and AR mRNA reduced in SS males. CONCLUSION: These results indicate SS and C males show differences in gene expression within brain regions controlling sexual behavior.

Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.

ATP is actively maintained at high concentrations in cancerous tissues, where it promotes a malignant phenotype through P2 receptors. In this study, we first evaluated the effect of extracellular ATP depletion with apyrase in SKOV-3, a cell line derived from metastatic ovarian carcinoma. We observed a decrease in cell migration and an increase in transepithelial electrical resistance and cell markers, suggesting a role in maintaining a mesenchymal phenotype. To identify the P2 receptor that mediated the effects of ATP, we compared the transcript levels of some P2 receptors and found that P2RX7 is three-fold higher in SKOV-3 cells than in a healthy cell line, namely HOSE6-3 (from human ovarian surface epithelium). Through bioinformatic analysis, we identified a higher expression of the P2RX7 transcript in metastatic tissues than in primary tumors; thus, P2X7 seems to be a promising effector for the malignant phenotype. Subsequently, we demonstrated the presence and functionality of the P2X7 receptor in SKOV-3 cells and showed through pharmacological approaches that its activity promotes cell migration and contributes to maintaining a mesenchymal phenotype. P2X7 activation using BzATP increased cell migration and abolished E-cadherin expression. On the other hand, a series of P2X7 receptor antagonists (A438079, BBG and OxATP) decreased cell migration. We used a CRISPR-based knock-out system directed to P2RX7. According to the results of our wound-healing assay, SKOV3-P2X7KO cells lacked receptor-mediated calcium mobilization and decreased migration. Altogether, these data let us propose that P2X7 receptor is a regulator for cancer cell migration and thus a potential drug target.

Purinergic Activation of Store-Operated Calcium Entry (SOCE) Regulates Cell Migration in Metastatic Ovarian Cancer Cells.

Store-operated calcium entry (SOCE) is an important process in calcium signaling. Its role in physiological and pathological events is well recognized. However, in cancerous systems, the importance of SOCE in relation to the degree of cancer aggressiveness, as well as its regulation by ligands such as purinergic molecules, are not well documented. This study aimed to characterize a differential effect of the P2Y2 receptor (promoted by UTP of 10 µM and inhibited by ARC118925XX of 1 µM) on intracellular calcium response between metastatic (SKOV-3) and non-metastatic (CAOV-3) ovarian cell lines in conditions of normal (1.5 mM) and zero extracellular calcium concentration. The sustained calcium influx observed exclusively in SKOV-3 cells was associated with the presence of SOCE (promoted by thapsigargin (74.81 ± 0.94 ΔF) and sensitive to 2-APB (20.60 ± 0.85 ΔF)), whereas its absence in CAOV-3 cells (26.2 ± 6.1 ΔF) was correlated with a low expression of ORAI1. The relevance of SOCE in metastatic SKOV-3 cells was further corroborated when 2-APB significantly inhibited (40.4 ± 2.8% of covered area) UTP-induced cell migration (54.6 ± 3.7% of covered area). In conclusion, our data suggest that SOCE activation elicited by the P2Y2 receptor is involved in the aggressiveness of ovarian cancer cells.

Bean Leaves Ameliorate Lipotoxicity in Fatty Liver Disease.

Bioactive compounds in plant-based food have protective effects against metabolic alterations, including non-alcoholic fatty liver disease (NAFLD). Bean leaves are widely cultivated in the world and are a source of dietary fiber and polyphenols. High fat/high fructose diet animal models promote deleterious effects in adipose and non-adipose tissues (lipotoxicity), leading to obesity and its comorbidities. Short-term supplementation of bean leaves exhibited anti-diabetic, anti-hyperlipidemic, and anti-obesity effects in high-fat/high-fructose diet animal models. This study aimed to evaluate the effect of bean leaves supplementation in the prevention of lipotoxicity in NAFLD and contribute to elucidating the possible mechanism involved for a longer period of time. During thirteen weeks, male Wistar rats (n = 9/group) were fed with: (1) S: Rodent Laboratory Chow 5001® (RLC); (2) SBL: 90% RLC+ 10% dry bean leaves; (3) H: high-fat/high-fructose diet; (4) HBL: H+ 10% of dry bean leaves. Overall, a HBL diet enhanced impaired glucose tolerance and ameliorated obesity, risk factors in NAFLD development. Additionally, bean leaves exerted antioxidant (↑serum GSH) and anti-inflammatory (↓mRNA TNFα in the liver) effects, prevented hepatic fat accumulation by enhanced ↑mRNA PPARα (ß oxidation), and enhanced lipid peroxidation (↓liver MDA). These findings suggest that bean leaves ameliorated hepatic lipotoxicity derived from the consumption of a deleterious diet.

In vitro conversion of ß-carotene to retinal in bovine rumen fluid by a recombinant ß-carotene- 15, 15'-monooxygenase.

Pasture-fed cattle yield carcasses with yellow fat; consumers often reject the resulting meat products because they assume they come from old and/or culled animals. Recombinant bacteria expressing beta-carotene 15, 15'-monooxygenase, introduced into the rumen of the animal, might help to reduce the coloration since this enzyme converts carotene to retinal, thereby eliminating the source of yellowness. The goal of this work was to evaluate the effect of a recombinant beta-carotene 15, 15'-monooxygenase (BCMO1) from Gallus gallus, expressed in Escherichia coli. The genetically modified microbe was introduced into ruminal fluid, and carotene conversion to retinal was measured. Under optimum conditions the enzyme produced 6.8 nmol of retinal per 1 mg of protein in 1 hour at 37 °C. The data on in vitro digestibility in ruminal fluid showed no differences in beta-carotene breakdown or in retinal production (p > 0.1) between E. coli with pBAD vector alone and E. coli with pBAD/BCMO1. The pBAD/BCMO1 plasmid was stable in E. coli for 750 generations. These results indicate that the protein did not break beta-carotene into retinal in ruminal fluid, perhaps due to its location in the periplasmic space in E. coli. Future research must consider strategies to release the enzyme into the rumen environment.

The glial cell's role in antinociceptive differential effects of oxytocin upon female and male rats.

BACKGROUND: Sex plays a crucial role in pain processing and response to analgesic drugs. Indeed, spinal glia seems to be significant in the sexual dimorphism observed in the above effects. Recently, studies have associated oxytocin with antinociceptive effects, but these have been mainly performed in male animals; consequently, the influence of sex has been poorly explored. METHODS: Using a model of spinal nociception that produces pain through activation of the spinal glia, that is, intrathecal (i.t.) lipopolysaccharide (LPS) injection, we analysed the changes in the analgesic response to i.t. oxytocin in female and male rats by behavioural (punctate mechanical hypersensitivity), electrophysiological (unitary extracellular recordings of wide dynamic range [WDR] cells) and molecular biology (real-time PCR of proinflammatory genes) experiments. RESULTS: We found that LPS-induced hypersensitivity was longer in female (>96 h) than in male (≈4 h) rats. Besides, spinal oxytocin preferentially prevents the LPS-induced hypersensitivity in male rather than female rats. Indeed, LPS increases the spinal neuronal-evoked activity associated with the activation of peripheral Aδ- and C-fibres and post-discharge in males, whereas only C-fibre discharge was enhanced in females. The electrophysiological data correlate with the fact that spinal oxytocin only prevented TNF-α and IL-1ß synthesis in male rats. CONCLUSIONS: Therefore, these data suggest that oxytocin-mediated analgesia depends on a sexual dimorphism involving activation of the spinal glia. These results reinforced the idea that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men. SIGNIFICANCE STATEMENT: Oxytocin is a molecule that emerges as a potent analgesic in preclinical and clinical studies. We investigated the contribution of glia to the response of oxytocin-induced analgesia and how sex influences in this response show that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men.

Chitosan Nanoparticles Containing Lipoic Acid with Antioxidant Properties as a Potential Nutritional Supplement.

The addition of the antioxidant α-lipoic acid (ALA) to a balanced diet might be crucial for the prevention of comorbidities such as cardiovascular diseases, diabetes, and obesity. Due to its low half-life and instability under stomach-like conditions, α-lipoic acid was encapsulated into chitosan nanoparticles (Ch-NPs). The resulting chitosan nanoparticles containing 20% w/w ALA (Ch-ALA-NPs) with an average diameter of 44 nm demonstrated antioxidant activity and stability under stomach-like conditions for up to 3 h. Furthermore, fluorescent Ch-ALA-NPs were effectively internalized into 3T3-L1 fibroblasts and were able to cross the intestinal barrier, as evidenced by everted intestine in vitro experiments. Thus, chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.

Conserved and divergent expression dynamics during early patterning of the telencephalon in mouse and chick embryos.

The mammalian and the avian telencephalon are nearly indistinguishable at early embryonic vesicle stages but differ substantially in form and function at their adult stage. We sequenced and analyzed RNA populations present in mouse and chick during the early stages of embryonic telencephalon to understand conserved and lineage-specific developmental differences. We found approximately 3000 genes that orchestrate telencephalon development. Many chromatin-associated epigenetic and transcription regulators show high expression in both species and some show species-specific expression dynamics. Interestingly, previous studies associated them to autism, intellectual disabilities, and mental retardation supporting a causal link between their impaired functions during telencephalon development and brain dysfunction. Strikingly, the conserved up-regulated genes were differentially enriched in ontologies related to development or functions of the adult brain. Moreover, a differential enrichment of distinct repertoires of transcription factor binding motifs in their upstream promoter regions suggest a species-specific regulation of the various gene groups identified. Overall, our results reveal that the ontogenetic divergences between the mouse and chick telencephalon result from subtle differences in the regulation of common patterning signaling cascades and regulatory networks unique to each species at their very early stages of development.

MCF-7 breast carcinoma cells express ryanodine receptor type 1: functional characterization and subcellular localization.

Breast carcinoma-derived MCF-7 cells are frequently used in biomedical research. However, few reports exist regarding the characterization of signaling mechanisms in these cancerous cells involved in intracellular Ca(2+) dynamics. Consequently, the aim of these experiments was to characterize the ryanodine receptor/Ca(2+) release channel (RyR) present in MCF-7 cells. Ryanodine (100 nM), cADPR (5 microM), and caffeine (10 mM) promoted cytoplasmic Ca(2+) mobilization; in contrast, ryanodine at inhibitory concentration (100 microM) decreased the basal Ca(2+) level. Fluorescent probes demonstrated that RyR is located mainly in endomembranes. Some degree of co-localization with inositol trisphosphate receptor (IP(3)R) was observed, whereas coincidence with thapsigargin-sensitive Ca(2+)-ATPase (SERCA) was more limited. Molecular cloning resulted in the detection exclusively of RyR isoform 1. For the first time, it is shown that MCF-7 cells express functional RyR.

Paced mating increases the expression of µ opioid receptors in the ventromedial hypothalamus of male rats.

Paced mating induces a positive affective state which is blocked by naloxone, an opioid antagonist. Opioids are released in the medial preoptic area (mPOA) and other brain regions during sexual behavior and µ opioid receptors (MOR) are activated in males that copulate until ejaculation. The aim of the present study was to determine if paced mating increases the expression of MOR in areas involved in the control of sexual behavior in male rats. Sexually experienced rats were assigned to one of the following groups: Paced, males that paced the sexual interaction; Non-Paced, males that did not pace the sexual interaction; Control, males were able to hear, see and smell a sexually receptive female, but no physical contact was possible. Males were sacrificed 4, 8 and 12 h after the behavioral tests. The mPOA, ventromedial hypothalamus (VMH), amygdala, olfactory bulbs (OB) and cortex were dissected and expression of MOR was determined by qPCR. In the VMH, the expression of MOR increased in the Paced group compared to the Non-Paced and Control groups at 4 h. No significant differences were found in the mPOA and the amygdala regardless of the time of sacrifice. In the OB and cortex, expression of MOR was not detectable. Interestingly, we found that the expression of MOR varied at the different times of sacrifice. In conclusion, our results show that the expression of MOR increased in the ventromedial hypothalamus after paced mating and it also varied in different brain areas depending on the time of the day.

A potential role for beta-carotene in avian embryonic development.

Vitamin A is essential for vertebrate embryonic development; dietary carotenoids are the primary source of vitamin A since animals cannot synthesize it de novo. To study the role of beta-carotene during embryonic development, we analyzed in chick embryos the expression of beta,beta-carotene 15,15'-oxygenase (beta-oxy) which cleaves beta-carotene to produce two molecules of retinal. Beta-oxy transcripts were detected in one-and-a-half- to five-day-old embryo homogenates and in situ hybridization in five-day-old embryos, revealing their presence in tissues including the central nervous system, lungs, limbs, and cardiovascular system. Moreover, we detected beta-oxy enzymatic activity in extracts from five-day-old embryos as well as small amounts of beta-carotene in the egg yolk. These results indicate that beta-oxy is present during early developmental stages, raising the possibility that yolk-stored beta-carotene is utilized as a source of vitamin A. Thus, our results suggest that beta-carotene could play an important role in early avian embryonic development as a local source of vitamin A in specific tissues.

Consumption of Ocimum sanctum L. and Citrus paradisi infusions modulates lipid metabolism and insulin resistance in obese rats.

A high saturated fat and fructose diet leads to metabolic disorders through dysregulation of genes involved in lipid metabolism. Consumption of plant infusions reduces these obesity alterations, but the precise mechanism remains unclear. In this study, we investigated the effect and the possible mechanism of Ocimum sanctum L. (OS) and Citrus paradisi (CP) infusions in diet-induced obese rats. CP and OS infusions suppressed hepatic tissue fat accumulation, and significantly down-regulated the mRNA levels of two hepatic lipogenesis genes: sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FAS) compared with the obese control. Treatment with these infusions up-regulated the hepatic expression of mRNA related to mitochondrial fatty acid uptake: peroxisome proliferator activated receptor alpha (PPARα) and the expression of carnitine palmitoyl-transferase 1a (CPT1a). Both infusions improved insulin resistance, with OS showing the major effect. Consumption of these infusions reduces the damage caused by free radicals, protecting hepatic lipids and proteins. Additionally, plant infusions increase activity of hepatic enzymes: glutathione S-transferase (GST), glutathione peroxidase (GPX), and catalase (CAT). Our results suggest that the effects of CP and OS infusions on lipid metabolism are related to the down-regulation of genes involved in lipogenesis, particularly for OS, and to the increase in lipid ß-oxidation, especially for CP infusion. In conclusion, the consumption of these plant infusions is a feasible adjuvant therapy for metabolic changes induced by obesity.

Repeated exposure to the herbicide atrazine alters locomotor activity and the nigrostriatal dopaminergic system of the albino rat.

Atrazine (ATR) is used as a pre- and post-emergent herbicide; although banned in several countries of the European Community, it is still used extensively around the world. A recent study in rats has shown that chronic, daily exposure to 10 mg ATR/kg BW causes hyperactivity, disrupts motor coordination and learning of behavioral tasks, and decreases dopamine levels in the brain. In order to evaluate the short-term effect of ATR exposure on locomotor activity, monoamine markers, and antioxidants, adult male Sprague-Dawley rats received six IP injections of 100 mg ATR/kg BW or vehicle over two weeks. After every ATR injection we found hypoactivity that lasted up to five days, and it was accompanied by reductions in levels of striatal DA, DOPAC, and HVA without any alteration in the striatal expression of the mRNAs for Mn-SOD, Trx-1, DAR-D(1), or DAR-D(2). In contrast, in the nucleus accumbens no changes in monoamine markers were observed, and a down-regulation of Trx-1 expression was detected shortly after the ATR treatment. Moreover, in the ventral midbrain, we found that ATR induced a down-regulation of mRNA for Th and DAT, but it increased VMAT2 mRNA expression. Decreases of monoamine levels and of locomotor activity disappeared three months after ATR treatment; however, an amphetamine challenge (1 mg/kg) given two months after the ATR treatment resulted in a significant stimulation in the exposed group, revealing hidden effects of ATR on dopaminergic systems. These results indicate that ATR exposure differentially modifies the dopaminergic systems, and these modifications may underlie the behavioral changes observed.

Evaluation of plasmid permanence in transfected cells after transplantation into the rat brain.

Plasmid retention after long-term transplantation has been one of the major technical limitations for transplantation studies. This study describes the use of a modified protocol of Hirt and a SYBR Green-based quantitative real-time PCR (qPCR) to recover and quantify a vector containing a specific transgene in transfected cells after brain transplantation. We compared various methods for sample processing and recovery of extrachromosomal DNA suitable for qPCR. The modified protocol of Hirt was the most reliable for optimal plasmid recovery from transplanted tissue with minimal loss of plasmid DNA compared to a commercial kit or TRIzol(®) protocols. The PCR protocol for plasmid and transgene detection included the design of two highly specific primer sets to detect the sequence for the human glutamate decarboxylase 1 (hGAD(67)) transgene by SYBR Green-based qPCR, and to confirm the presence of vector pREP10 hGAD(67) by end-point PCR. We used a standard curve constructed from serial dilutions of pure plasmid pREP10 hGAD(67) as reference in qPCR experiments to determine the number of plasmid copies recovered from cultured cells and tissue samples after Hirt extraction. Then, plasmid permanence was evaluated in transplanted tissues after different time intervals, and plasmid loss in the tissue of interest was found to be time dependent. In this study we describe an easy, highly specific, low-cost, and reliable method for plasmid recovery and quantification of a transgene of interest in long-term brain transplantation studies; use of this method may be extended to other transplantation models.