Designing primers and evaluation of the efficiency of propidium monoazide – Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius

AbstractThe purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4–5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable

Paternal Tobacco Smoke Correlated to Offspring Asthma and Prenatal Epigenetic Programming

Rationale: Little is known about effects of paternal tobacco smoke (PTS) on the offspring’s asthma and its prenatal epigenetic programming.Objective: To investigate whether PTS exposure was associated with the offspring’s asthma and correlated to epigenetic CG methylation of potential tobacco-related immune genes: LMO2, GSTM1 or/and IL-10 genes.Measurements and Main Results: In a birth cohort of 1,629 newborns, we measured exposure rates of PTS (23%) and maternal tobacco smoke (MTS, 0.2%), cord blood DNA methylation, infant respiratory tract infection, childhood DNA methylation, and childhood allergic diseases. Infants with prenatal PTS exposure had a significantly higher risk of asthma by the age of 6 than those without (p = 0.026). The PTS exposure doses at 0, <20, and ≧20 cigarettes per day were significantly associated with the trend of childhood asthma and the increase of LMO2-E148 (p = 0.006), and IL10_P325 (p = 0.008) CG methylation. The combination of higher CG methylation levels of LMO2_E148, IL10_P325, and GSTM1_P266 corresponded to the highest risk of asthma by 43.48%, compared to other combinations (16.67–23.08%) in the 3-way multi-factor dimensionality reduction (MDR) analysis. The LMO2_P794 and GSTM1_P266 CG methylation levels at age 0 were significantly correlated to those at age of 6.Conclusions: Prenatal PTS exposure increases CG methylation contents of immune genes, such as LMO2 and IL-10, which significantly retained from newborn stage to 6 years of age and correlated to development of childhood asthma. Modulation of the LMO2 and IL-10 CG methylation and/or their gene expression may provide a regimen for early prevention of PTS-associated childhood asthma.Descriptor number: 1.10 Asthma Mediators.Scientific Knowledge on the Subject: It has been better known that maternal tobacco smoke (MTS) has an impact on the offspring’s asthma via epigenetic modification. Little is known about effects of paternal tobacco smoke (PTS) on the offspring’s asthma and its prenatal epigenetic programming.What This Study Adds to the Field: Prenatal tobacco smoke (PTS) can program epigenetic modifications in certain genes, such as LMO2 and IL-10, and that these modifications are correlated to childhood asthma development. The higher the PTS exposure dose the higher the CG methylation levels are found. The combination of higher CG methylation levels of LMO2_E148, IL10_P325 and GSTM1_P266 corresponded to the highest risk of asthma. Measuring the DNA methylation levels of certain genes might help to predict high-risk populations for childhood asthma and provide a potential target to prevent the development of childhood asthma

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Pharmacological and Genetic Studies of Effort-Related Decision Making Using Rodent Models: Roles of Dopamine in Motivated Behavior

The major objective of the present research was to characterize the underlying mechanisms of effort-related aspects of motivation using pharmacological and genetic approaches in rodent subjects. Effort-related decision making tasks have been widely used to assess motivated behavior in human and animals. However, human studies often yield mixed results due to less controllable genetic heterogeneity. Additionally, considering the importance of developing clinical interventions for human psychiatric disorders, there is a need to establish animal models that are useful for understanding the mechanisms of motivational symptoms. This dissertation consists of five sets of experiments which aimed to investigate the roles of dopamine (DA) and adenosine in motivated behavior in rats, and to develop novel touchscreen procedures using transgenic mouse models and pharmacological interventions to assess DA related genes and DA transmission in regulating effort-related decision making. Exp 1 demonstrated that the administration of DA depleting agent tetrabenazine (TBZ) and the DA D2 receptor antagonist haloperidol increased elasticity of demand for food in rats. In addition to DA, Exp 2 showed the ability of the highly selective adenosine A2A receptor antagonist preladenant to reverse the motivation effects induced by TBZ in a rat effort-related choice task. While the neural mechanisms underlying effort-related choice behavior have been widely studied in rats, fewer studies have been performed in mice. Exp 3 established mouse effort-related choice procedures by using Bussay-Sakisda touchscreen chambers, and evaluated the effects of haloperidol on CD1 mice responding on the touchscreen paradigm. Using the developed touchscreen paradigm, Exp 4 indicated that transgenic mice carrying humanized catechol-o-methyltransferase gene valine allele was associated with motivational impairments. Moreover, Exp 5 studied the effects of TBZ on C57BL6 mice tested on the touchscreen procedures, and determined the effects of TBZ on striatal DA levels with a tissue assay and high performance liquid chromatography. This research could lead to a greater understanding of the neurochemical and genetic mechanisms underlying effort-based aspects of motivation, and these models could serve as a platform for assessment of novel strategies for the treatment of motivational dysfunctions in human psychiatric and neurological patients

Investigation of neural mechanisms of risky choice behavior in the rat

「風險決策」行為非常普遍的存在於吾人之日常生活中，而選項所帶來的風險和獎勵是吾人進行決策時的重要考量因素。風險選擇的適當與否，對於個體的生存扮演著相當重要的角色。在以往的文獻中，對於決策的行為歷程已有所關注及探討，但對於風險選擇行為的神經生理機制迄今未明。本研究藉由大白鼠於T字迷津中，選擇確定之低酬賞或高不確定性之高酬賞的行為表現，進行風險選擇行為的探討。本研究中以兩項主要實驗，探討風險選擇行為之神經行為機制。實驗1a中，確定之低酬賞端固定呈現1顆食物粒，而高不確定性之高酬賞端則同時操弄酬賞物機率(50%、25%及12.5%)以及酬賞物的量(2、4及8顆)，以系統性地檢驗期望值(0.5、1和2)於此風險選擇行為中扮演的角色。行為結果顯示當風險較低時，大白鼠會選擇高不確定性之高酬賞端；而風險較高時，則轉為選擇確定之低酬賞端。實驗1b中，系統性地施打不同劑量之安非他命，探討多巴胺系統在此風險選擇行為中之機制。實驗結果顯示施打安非他命後，大白鼠表現出相對地追求風險之行為，亦即選擇高不確定之高酬賞端之比例顯著高於控制組。實驗2中，藉由毀除大腦特定部位(依核、背外側之紋狀體、眶前額皮質、內側之前額皮質)，檢驗風險選擇行為之神經基礎。毀除後之結果顯示，僅有依核受到毀除之大白鼠表現出相對地趨避風險之選擇行為。綜合以上結果，本研究建立之風險選擇行為與多巴胺有關，而依核在此行為歷程中扮演重要的調節角色。Many decisions people make every day involve uncertainty where both risks and rewards associated with each option need to be considered. Behavioral performance associated to risk-based choice appears wildly over the lifespan, and the fitness of risky choice behavior plays an important role in individual survival. Despite a growing body of research has focused to investigate the neurobiology of decision making, little is known about the neurobehavioral mechanisms of risky choice behavior. Based on a pilot work, this study used a T-maze to study decision under a probability-based risk in the rat. The subject was assessed on making choice to obtain either a large reward associated with risk of non-reward “empty” or a small reward ensured for every entry. Two experiments were conducted in this project to investigate neurobehavioral mechanisms of probabilistic risky choice behavior. In Experiment 1a, probabilistic risky choice behavior was systemically assessed under three expected values (0.5, 1.0, and 2.0) by manipulating the probabilities of reward presence (50%, 25%, and 12.5%) and the reward magnitude (2, 4, or 8 pellets) in the probabilistic high reward (PHR) arm. Behavioral data showed that the subject chose the probabilistic high reward in a lower risk condition but would shift to the choice of certain low reward (CLR) as the risk is increased. In Experiment 1b, the dose effects of amphetamine on this probabilistic risky choice task was tested to verify whether the dopaminergic mechanism was involved. Amphetamine, presumably activating brain dopamine systems, produced a relatively risk-seeking effect on the present behavioral task. In Experiment 2, the excitoneurotoxic lesion was conducted in the nucleus accumbens, the dorsolateral striatum, the orbitofrontal cortex, and the medial prefrontal cortex to examine the neural substrates for this probabilistic risky choice behavior. The results showed that the lesion of the nucleus accumbens significantly produced a relatively risk-averse effect on the present behavioral task, as compared to the lesions made on the other three brain areas. In conclusion, the probabilistic risky choice behavior established in the present study is dopamine dependent. And, the nucleus accumbens plays a major role of mediating this behavioral processing

Lisdexamfetamine suppresses instrumental and consummatory behaviorssupported by foods with varying degrees of palatability: Exploration of abinge-like eating model

Diets high in sugar or fat are associated with multiple health conditions, including binge eating disorder (BED). BED affects approximately 2% of the US adult population, and occurs more frequently in females. It is important to develop animal models of palatable food consumption and food seeking that may have relevance for BED and other conditions associated with excessive food intake. The catecholamine uptake blocker and d-amphetamine prodrug lisdexamfetamine is used to treat BED. The present experiments studied the effect of lisdexamfetamine on food intake and food-reinforced effort-based choice in female Wistar rats. Three groups of rats received different food exposure conditions in the home cage randomly spread over several weeks: a chocolate exposure group (CE; brief access of chocolate and additional lab chow, n = 15), a lab chow exposure (LChE) group given additional access to lab chow (n = 8), and a third group given empty food dishes (n = 7). In tests of food intake under non-restricted conditions, lisdexamfetamine (0.1875–1.5 mg/kg IP) significantly reduced intake of both chocolate and chow in the CE group. In the LChE group, there was a trend towards reduced chow intake induced by lisdexamfetamine. All rats were trained on a Progressive Ratio/chow feeding choice task, in which they had a choice between working for high carbohydrate chocolate flavored pellets by lever pressing vs. approaching and consuming a concurrently available lab chow. The LChE group and the empty food dish group were combined to create one control group (n = 15). There was a significant overall dose-related suppressive effect of lisdexamfetamine on lever pressing but no group difference, and no dose x group interaction. Lisdexamfetamine significantly decreased chow intake in the CE group, but not in the control group. In conclusion, lisdexamfetamine affected both food intake and food-reinforced operant behavior, with larger effects seen in the group exposed to chocolate

Dopamine, Effort-Based Choice, and Behavioral Economics: Basic and Translational Research

Operant behavior is not only regulated by factors related to the quality or quantity of reinforcement, but also by the work requirements inherent in performing instrumental actions. Moreover, organisms often make effort-related decisions involving economic choices such as cost/benefit analyses. Effort-based decision making is studied using behavioral procedures that offer choices between high-effort options leading to relatively preferred reinforcers vs. low effort/low reward choices. Several neural systems, including the mesolimbic dopamine (DA) system and other brain circuits, are involved in regulating effort-related aspects of motivation. Considerable evidence indicates that mesolimbic DA transmission exerts a bi-directional control over exertion of effort on instrumental behavior tasks. Interference with DA transmission produces a low-effort bias in animals tested on effort-based choice tasks, while increasing DA transmission with drugs such as DA transport blockers tends to enhance selection of high-effort options. The results from these pharmacology studies are corroborated by the findings from recent articles using optogenetic, chemogenetic and physiological techniques. In addition to providing important information about the neural regulation of motivated behavior, effort-based choice tasks are useful for developing animal models of some of the motivational symptoms that are seen in people with various psychiatric and neurological disorders (e.g., depression, schizophrenia, Parkinson’s disease). Studies of effort-based decision making may ultimately contribute to the development of novel drug treatments for motivational dysfunction