A receptor antagonist picrotoxin could effectively ameliorate several behavioral defects of GAT1 KO mice. These results identified a novel function of GAT1, and indicated that the elevated ambient GABA contributed critically to the pathogenesis of schizophrenia. Furthermore, several commonly used antipsychotic drugs were effective in treating the locomotor hyperactivity in GAT1 KO mice, suggesting the utility of GAT1 KO mice as an alternative animal model for studying schizophrenia pathogenesis and developing new antipsychotic drugs.The mechanism underlying the pathogenesis of schizophrenia remains poorly understood. The hyper-dopamine and hypo-NMDA receptor hypotheses have been the most enduring ideas. Recently, emerging evidence implicates alterations of the major inhibitory system, GABAergic neurotransmission in the schizophrenic patients. However, the pathophysiological role of GABAergic system in schizophrenia still remains dubious. In this study, we took advantage of GABA transporter 1 (GAT1) knockout (KO) mouse, a unique animal model with elevated ambient GABA, to study the schizophrenia-related behavioral abnormalities. We found that GAT1 KO mice displayed multiple behavioral abnormalities related to schizophrenic positive, negative and cognitive symptoms. Moreover, GAT1 deficiency did not change the striatal dopamine levels, but significantly enhanced the tonic GABA currents in prefrontal cortex. The GABA A receptors (GABAARs) in the prefrontal cortex of human subjects with schizophrenia Schizophrenia is a highly debilitating mental disorder that affects approximately 1% of the world's population, which pathogenesis mechanisms remain unclear. Traditionally, the hyper-dopamine hypothesis The GAT1 is primarily responsible for the removal of GABA from the synaptic cleft and termination of GABAergic neurotransmission. It belongs to high-affinity, sodium- and chloride-dependent GABA transporters, and is predominantly abundant in GABAergic neurons A receptor antagonist picrotoxin could effectively ameliorate several behavioral defects of GAT1 KO mice. These results underscore the significance of elevated ambient GABA in the pathogenesis and treatment of schizophrenia. Moreover, we found that several commonly used antipsychotic drugs were effective in treating the locomotor hyperactivity in GAT1 KO mice, suggesting the utility of GAT1 KO mice as an alternative animal model for studying schizophrenia pathogenesis and testing new antipsychotic drugs.In this study, we found that GAT1 KO mice displayed multiple schizophrenia-like behaviors, suggesting that GAT1 downregulation may be a pathogenic mechanism, but not a simple compensatory change. Moreover, the striatal dopamine levels were unchanged in GAT1 KO mice, but the tonic GABA currents in prefrontal cortex were significantly increased. The GABAad libitum except during tests. In all experiments, the investigators were blind to the genotype of mice. The experiments were performed on the mice in a randomized order.The care and use of animals in these experiments followed the guidelines of, and the protocols were approved by, the Institutional Animals Care and Use Committee of the Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The mGAT1 KO strain was used in this study. The details of the targeting construct, homologous recombination, and genotyping were described previously in vivo microdialysis, in vitro electrophysiology and Western blot tests. For observation-based behavior tests, i.e. open field tests without drug administration, novel object recognition, nesting behavior, social interaction, and Y-maze spontaneous alternation, mice were used repeatedly with a certain time intervals.In the following experiments, mice were only used for one time: the drug treated experiments, prepulse inhibition, latent inhibition, Morris water maze, Adult WT and GAT1 KO male mice (3–5 months old littermates) were used in most of the behavioral tests and 4–5 weeks old mice (young mice) were used to investigate the emergence time of the schizophrenia-like phenotypes. For the drug treatment in behavioral tests, mice were injected intraperitoneally (i.p.) in a volume of 10 ml/kg body weight.Mice were placed in the center of a square Plexiglas open field apparatus (40×40×35 cm) and allowed to freely explore for 30 min. The same procedure was repeated once daily for 4 consecutive days. Total distance traveled was quantified using the Ethovision videotracking system . To test the stimulant effects of NMDAR antagonist MK801 and phencyclidine (PCP), mice were injected i.p. with drug after 30-min acclimation in the open field, and then immediately tested for another 30 min. To test the effects of antipsychotic drugs and picrotoxin (PTX), mice were not acclimated to the open field but directly injected i.p. with drug 30 min before the test.Mice were individually habituated to an open field box (50×50×35 cm) before test. In the first day, two different objects (A and B) were used as acute novelty and placed into two distinct quadrants of the open field and each mouse was allowed to explore for 5 min. The object recognition was tested 24 h later. The mice were placed back into the same box, in which one of the familiar objects (A) was replaced by a novel object (C), and allowed to explore freely for 5 min. The time spent in the proximity of the object (the quadrant with object) was measured using Ethovision.Ten pieces of the nesting material, made of cotton fiber, was introduced into an empty cage. Then, a mouse was placed in the cage with food and water for an overnight period. Pictures of the nests were taken by a digital camera and exported into a computer. The number of cotton particles was counted for each cage.Social interaction was measured by placing the mouse in a clean empty cage for 60-min acclimation and then introducing an unfamiliar young male C57BL/6J mouse (3-week old) for 10 min. Sniffing, nosing, grooming, and crawling over each other are considered social interest behavior. The duration of social interest behaviors carried out by the resident mouse was recorded by an experienced observer who remained blind to the animal's genotype.A startle reflex measurement system was used  in the PPI test. During the test, the animal was confined to the holder. Background noise was set at 65 dB. Five types of trials were used. Pulse alone trials (P) consisted of a single white noise burst . The prepulse+pulse trials  consisting of a prepulse of noise  followed by a startling pulse  100 ms after prepulse onset. No-stimulus (NS) trials consisted of background noise only. Sessions were structured as follows: (1) 5-min acclimation at background noise level; (2) five P trials; (3) 10 blocks each containing all five trials  in pseudorandom order; and (4) five P trials. Inter-trial intervals were distributed between 12 and 30 s. The force intensity for each trial was recorded as the startle level. Startle magnitude in this formula was calculated as the average response to all of the pulse alone trials, excluding the first and last blocks of five pulse alone trials. The percentage PPI induced by each prepulse intensity was calculated as [1-/]×100%.Latent inhibition was measured by cued fear conditioning test. The fear conditioning test was performed by a Near Infrared (NIR) Video Fear Conditioning System . During training session, each mouse was placed into the conditioning chamber for 3 min. The mice were divided into two groups: pre-exposed (P) group and non pre-exposed (NP) group. The P group received 40 white noise tones , whereas the NP group received no stimulus during an equivalent period. Immediately after the tone pre-exposure or the exposure to the chamber, tone-shock pairs consisting of a 5 s tone co-terminating with a 2 s foot shock at 0.70 mA were delivered to both groups with a 30 s inter-stimulus interval. During the retention test 24 h later, the mice were put in a white Plexiglas chamber different from the conditioning chamber, and after 180 s, a 180 s tone was delivered to measure cued freezing.The Morris water maze consisted of a circular pool  filled with water at 24–26°C to a depth of 20 cm. The water surface was covered with floating white resin beads. Yellow curtains were drawn around the pool (50 cm from the pool periphery) and contained distinctive visual marks that served as distal cues. Before training, a 60 s free swim trial without the platform was run. In this test, each mouse was given four consecutive 60-sec training trials  every day for 4 d. A submerged  platform was fixed in the centre of a quadrant for all four trials each day, but the platform location was changed between days. Each mouse was allowed to stay on the platform for 30 s. Swimming paths for training session and probe test were monitored using an automatic tracking system. This system was used to record the swimming trace and calculate the latency to the platform.The Y-maze apparatus was made of Plexiglas and had three identical arms (40×10×15 cm) placed at 120° with respect to each other. Specific motifs were placed on the walls of each arm, thus allowing visual discrimination. Each mouse was placed at the end of one arm and allowed to explore the apparatus freely for 5 min, with the experimenter out of the animal's sight. Total entries were scored as an index of ambulatory activity in the Y-maze. Alternations were defined as successive entries into each of the three arms on overlapping triplet sets. Percentage spontaneous alternation performance (SAP) was defined as the ratio of actual  to possible  alternations×100. The alternate arm returns (AARs) were also scored for each animal. If an animal went, for example, from arm A to arm B and back to arm A, one AAR was recorded.In vivo microdialysis measurements of extracellular dopamine and metabolites were performed in freely moving mice in an identical manner to that described before 2, 1.2 mM MgCl2, 5.0 mM glucose, pH 7.4). Perfusion with ACSF started at 12 h after the probe insertion and lasted for 2 h at a rate of 2 µl/min for equilibrium. Afterward, dialysate samples were collected into polypropylene microcentrifuge vials at 20 min intervals. All samples were frozen at −70°C before they were analyzed. At the end of the experiments, the coronal sections of the brain were cut to verify the location of the probe. Dialysis samples were assayed for dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenylacetic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The levels of neurotransmitters in the samples were calculated by extrapolating the peak area from a standard curve.2/5% CO2) ACSF at 4°C. Slices were cut with a Leica VT1000S vibratome  and maintained at room temperature (23–25°C) in a holding chamber filled with oxygenated ACSF for at least 2 h, whereas slices for whole-cell recordings were initially incubated in warmed (32°C) ACSF for 30 min and then maintained at room temperature. Then a single slice was transferred to the recording chamber, where it was held between two nylon nets and continuously perfused with oxygenated ACSF (23–25°C) at a flow rate of 2–3 ml/min. The same ACSF was used in cutting, incubating and recording, and contained (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1 NaH2PO4, 1.3 MgSO4, 26.2 NaHCO3, and 11 D-glucose, saturated with 95% O2/5% CO2 (pH 7.4). The osmolarity of the ACSF was 310–320 mOsm/l. All electrophysiological recordings were performed at room temperature with an Axopatch-200B amplifier  at the sampling rate of 10 kHz and filtered at 5 kHz. Data were acquired and analyzed using a Digidata 1322A interface and Clampfit 9.0 software . Whole-cell recordings were made from the layer II/III pyramidal neurons of PFC. The neurons were visually identified using an upright microscope  equipped with differential interference contrast optics and an infrared camera. Patch pipettes were made from borosilicate glass (1.5 mm OD) with a micropipette puller . The internal pipette solution for voltage-clamp recording contained (in mM): 140 KCl, 5 NaCl, 2 MgATP, 0.3 NaGTP, 0.1 EGTA, 10 HEPES. The pH was adjusted to 7.2, and the osmolarity was 300–310 mOsm/l. To block action potentials, 2 mM QX-314 was added into the pipette solution. The resistance of the patch electrode filled with above internal solution was 3–5 MΩ. Under voltage-clamp conditions, the cells were held at −70 mV. Series resistances were usually 10–20 MΩ. To record inhibitory postsynaptic currents (IPSCs), 10 µM CNQX and 20 µM d-APV were added to the ACSF to block glutamatergic responses. Tonic GABAAR-mediated currents were examined by applying the selective GABAAR antagonist picrotoxin into the slice chamber in a final concentration of 100 µM. The tonic GABA current was measured as the outward shift in the holding current.Coronal slices (350 µm thick) containing prefrontal cortex (PFC) were prepared from 6–10 weeks old male WT or GAT1 KO littermate mice. After decapitation, the brain was removed and placed in oxygenated . After lysed on ice for 30 min, the lysates were centrifuged at 13,000 g for 15 min at 4°C. The supernatants were collected and separated on 10% SDS-PAGE gels with 4% stacking gels. Then the proteins were transferred to polyvinylidene difluoride filters and incubated overnight with anit-GABAAR α1 , anit-GABAAR γ2 , anit-GABAAR α2 , anit-GABAAR α5 , anit-GABAAR β , anit-GABAAR δ  and anti-GAPDH . Secondary antibodies conjugated with horseradish peroxidase were then incubated with the filters at room temperature (23–25°C) for 2 h. After that the filters were visualized in ECL solution using the ImageQuant LAS 4000 mini Molecular Imaging System  and their optic densities were analyzed via the Image J software .The PFC from WT or GAT1 KO littermate mice was dissected according to the mouse brain atlas and were homogenized in the lysis buffer contained 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 3 mM NaF, 1 mM β-glycerophosphate, 1 mM Nat-test. All statistical analysis was performed using Origin 7.0 .All drugs and chemicals in these experiments were purchased from Sigma . In the experiments with cortical slices, drugs were applied to the bathing medium. All the data were shown as the mean ± S.E.M., with statistical significance assessed by two-way AVONA and unpaired Student's p<0.01, t-test, p>0.1, ANOVA, p>0.1, ANOVA, p<0.001, p<0.001, ANOVA, Schizophrenia is diagnosed on the basis of characteristic clinical syndromes in human individuals, including positive, negative and cognitive symptoms p>0.1; 0.3 mg/kg vs saline, p<0.01, ANOVA, p = 0.03; 0.3 mg/kg vs saline, p<0.01; genotype×dose interaction, F = 3.1, p = 0.05, ANOVA, p<0.01; GAT1 KO, saline, 39.6±3.8 m, PCP, 129.3±10.8 m, F = 61.4, p<0.01, n = 6–7 for each group, ANOVA, p<0.01, ANOVA, Another feature of positive symptom in schizophrenia is the augmented response to psychotomimetic drugs, such as non-competitive NMDAR antagonists MK801 and PCP p<0.01; object B, WT, 48.3±9.5 s, KO, 89.0±8.8 s, F = 10.0, p<0.01; n = 12–13 for each group, ANOVA, p>0.1, ANOVA; p = 0.02, ANOVA, p>0.1, ANOVA, Then, we introduced two objects A and B into two distinct quadrants of the open field and counted the time spent near the objects by mice in a 5-min test. GAT1 KO mice spent significantly more time in the proximity of both objects than WT  = 14.6, p<0.01, t-test, p<0.01, t-test, Social behavioral tests in rodents are used to model negative symptoms of schizophrenia. Nesting is a common index of social behavior p>0.1, t-test, p<0.05, ANOVA, p<0.01, ANOVA, Prepulse inhibition is a common measure of sensorimotor gating, which is decreased in schizophrenic patients p<0.01, ANOVA, p<0.01, ANOVA, p>0.1, ANOVA, Latent inhibition is another measure for the attentional function, and is abnormal in schizophrenic patients p>0.1, t-test, p<0.001; genotype×trial interaction, F = 15.329, p<0.001; n = 10–11 mice×4 days×4 trials for each genotype, ANOVA, Cognitive impairment is an important feature in schizophrenia. In the previous study, we reported that GAT1 KO mice showed general long-term cognitive deficits in Morris water maze, passive avoidance and contextual fear conditioning tests p<0.01, t-test, p<0.01, p<0.01, t-test), further indicating the impaired working memory of GAT1 KO mice.Then, we examined the spontaneous alternation behavior in the Y-maze test, which is based on the animal's willingness to explore novel environmental stimuli and thought to reflect working memory performance. Consistent with the locomotor hyperactivity in open field, GAT1 KO mice exhibited more total arm entries in 5-min Y-maze tests than WT mice  = 13.5, p<0.01; risperidone, F = 96.6, p<0.01; n = 7–9 for each group, ANOVA, p<0.01; clozapine, F = 1.9, p = 0.09; risperidone, F = 13.2, p<0.01; n = 7–9 for each group, ANOVA, According to the above behavioral results, GAT1 KO mice displayed multiple behavioral abnormalities related to schizophrenic positive, negative and cognitive symptoms, suggesting that GAT1 KO mice may be a novel schizophrenia mouse model. Then we want to know whether commonly used antipsychotic drugs are effective in this mouse model. Here we chose a typical drug haloperidol and two atypical drugs clozapine and risperidone, and examined their effects on the locomotor hyperactivities of GAT1 KO mice. We found that in both WT and GAT1 KO mice, all three drugs could reduce the locomotor activity in a dose-dependent manner  = 48.0, p>0.1; HVA, p>0.1; 5-HIAA, p>0.1; n = 4–6 for each genotype, t-test, Increased dopaminergic transmission has been considered to be a mechanism in schizophrenia. Furthermore, the antipsychotic drugs used in A antagonist PTX (100 µM) failed to induce an obvious tonic current in WT slices  in the PFC of WT and GAT1 KO mice. All these subunits remained unchanged in the PFC of GAT1 KO mice compared to their WT littermates  = 0.86, p>0.1; AAR, F = 0.86, p>0.1; n = 7 for each group, ANOVA, The GABAergic inhibition is increased in the PFC and hippocampus, as well as cerebellum in the GAT1 KO mice. Thus, we want to know whether this increased GABAergic inhibition contributes to the schizophrenia-like behaviors. We used PTX to study the effect of GABAergic antagonism in GAT1 KO mice, and found that PTX at a dose below the threshold to induce seizure (3 mg/kg) could significantly alter locomotor activity in both WT and KO mice  = 18.8, 3 mg/kg vs vehicle, p<0.01, t-test, p<0.001, ANOVA, p<0.001, ANOVA, p<0.01, t-test, p<0.01, t-test, p<0.01, t-test, p<0.01, t-test, Schizophrenia is a neurodevelopmental disorder which is closely associated with environmental and developmental vulnerability factors In previous studies, GAT1 KO mice have been found to have several behavioral abnormalities, including altered responses to ethanol AR α2 subunit in the PFC in schizophrenic patients AR α2 subunit is predominantly expressed in the pyramidal neuron axon initial segments, and upregulated in schizophrenic patients ARs and decreased GABA reuptake. It has been supposed in several studies that GAD67 downregulation is the dominant mechanism in schizophrenia while the increase of GABAAR and the decrease of GAT1 are likely to be compensatory alterations AR α2 agonist could improve prefrontal cognitive function in schizophrenic patients AR α2 agonist may be more complicated in terms of producing inhibition. Furthermore, several atypical antipsychotics, such as clozapine and olanzapine, can antagonize GABAAR in the cortex and decrease extracellular GABA in the PFC Besides dopaminergic and glutamatergic hypotheses, GABAergic mechanism has attracted increasing attentions in the study of schizophrenic pathogenesis. Although genetic studies have yielded little in the study of GABAergic system in schizophrenia, postmortem human studies have found out GABAergic disturbances in the brain of schizophrenic patients. These changes include reduced mRNA level and expression of GAD67 and GAT1, and upregulation of GABAIn most neurological diseases, such as epilepsy, ischemia, chronic pain, also including schizophrenia, GABAergic disinhibition has been thought as a main pathogenic mechanism. However, several recent studies showed that tonic GABA inhibition was enhanced in epilepsy and ischemia, while reducing the increased tonic GABA inhibition was effective in the treatment of both diseases Neural oscillations play a fundamental role in coordinated activity during normal brain functioning As the major inhibitory system in the brain, GABAergic system has important functions in modulating glutamatergic and dopaminergic transmissions, which are largely involved in the pathogenesis of schizophrenia. For instance, hypofunction of NMDAR has been proposed as an underlying cause in schizophrenia pathophysiology The GABAergic efficacy can also modulate dopamine activity, but in a more complicated manner, depending on the intensity of GABA manipulation and pre-existing dopamine activity Animal models are useful tools in studying the pathogenesis and treatment in human diseases. Although several symptoms of schizophrenia are difficult to measure directly in rodents, various behavioral tests in rodents have been established with potential relevance to the symptoms of schizophrenia


The rice bean varieties had significant differences in total phenolic compounds (ranging from 123.09 ± 10.35 to 843.75 ± 30.15 μg/g), in TPC /g), in TFC (ranging from 55.95 ± 11.16 to 320.39 ± 31.77 mg catechin (CE)/g), in antioxidant activity (ranging from 39.87 ± 1.37 to 46.40 ± 2.18 μM·TE/g), in α-glucosidase inhibition activity (ranging from 44.32 ± 2.12 to 68.71 ± 2.19) and in advanced glycation end products formation inhibition activity (ranging from 34.11 ± 0.59 to 75.75 ± 0.33). This study is the first report on phytochemistry and biological activities in rice beans.Interest in edible beans as nutraceuticals is increasing. In the present study, the individual phenolic acids, the total phenolic content (TPC), the total flavonoid content (TFC), and the antioxidant and antidiabetic potential of 13 varieties of rice beans from China were investigated. Eight phenolic compounds (catechin, epicatechin,  Antioxidants refer to compounds possessing free radical-scavenging activity, transition metal-chelating activity, and/or singlet oxygen-quenching capacity ,2. SeverActing as a key enzyme for carbohydrate digestion, intestinal α-glucosidase is one of the glucosidases located at the epithelium of the small intestine. Alpha-Glucosidase has been recognised as a therapeutic target for modulation of postprandial hyperglycaemia, which is the earliest metabolic abnormality to occur in type II diabetes mellitus ,6. InhibVigna umbellata L.), known as climbing mountain bean, mambi bean and oriental bean, is native to Southeast Asia [Rice bean  radical, Folin-Ciocalteu reagent, rat intestinal acetone powder, bovine serum albumin (BSA), D-glucose and methylglyoxal (MGO) were all purchased from Sigma-Aldrich . All of the chemicals were of analytical grade and were obtained from Beijing Chemical Reagent . All of the analytical grade solvents for high performance liquid chromatography (HPLC) were purchased from Fisher Chemicals .Thirteen varieties of rice beans were provided by the Chinese National Genebank . Trolox, catechin, epicatechin, All dried samples were ground in a laboratory mill and passed through a sieve (80 mesh). Bean samples (10 g) were extracted twice in 100 mL of 70% ethanol for 2 h at room temperature. After vacuum filtration, the supernatants were combined and concentrated under reduced pressure in a rotary evaporator at 50 °C. After freeze-drying, the sample powder was stored at −20 °C until analysis. The biological activities of the rice bean were measured at a concentration of 10 mg/mL.2CO3 solution. The mixture was thoroughly mixed and allowed to stand for 60 min at room temperature before reading the absorbance at 765 nm . Quantification was performed with respect to the standard curve of gallic acid. The results were expressed as milligrams of gallic acid equivalent (GAE) per gram.TPC was measured using the Folin-Ciocalteu method as previously described by Zhou, Fang, Lü, Chen, Liu, and Ye  and modi2 solution. After 6 min, 0.15 mL of a 10% AlCl3 solution was added to the mixture and the solution was then allowed to stand for 6 min followed by the addition of 2 mL of a 4% NaOH solution. Water was immediately added to bring the final volume to 5 mL, and the mixture was then thoroughly mixed and allowed to stand for an additional 15 min before the absorbance was measured. The results were expressed as micrograms of catechin equivalent (CE) per gram.TFC was measured using the colorimetric method as previously described by Zou, Lu, and Wei  with somThe chromatographic system consisted of an ultra performance liquid chromatography system equipped with a Triple Quadrupole Mass Spectrometer (TQD) . The analytical column was a Waters ACQUITY UPLC BEH Shield RP18  column. The following mobile phase consisting of methanol (A) and 0.1% (v/v) formic acid in water (B) was used in a gradient elution: 0–5 min with 35–40% solution A; 5–8 min with 40–65% solution A; 8–10 min with 65–90% solution A; 10–11 min with 90% solution A; 11–13 min with 90–35% solution A; and 13–15 min with 35% solution A. The flow rate was 0.25 mL/min, and the injection volume was 10 μL. The column and autosampler were maintained at 35 °C and 10 °C, respectively. Identification and quantification of phenolic compounds were performed by comparing the retention times of authentic standards and the areas of external standards.The DPPH radical-scavenging activity was determined using the method reported by Yao, Sang, Zhou, and Ren . DPPH (1Acontrol − Asample)/Acontrol × 100%. The IC50 value was defined as the concentration of bean extracts required to inhibit 50% of the enzyme activity.The α-glucosidase inhibition activity was determined as previously described with slight modifications . Alpha -et al. [BSA-glucose and BSA-MGO models were used for the evaluation of the inhibition effect of the extracts on the formation of advanced glycation end products. The BSA-glucose assay was carried out according to the method reported by Peng et al. . BrieflyThe BSA-MGO assay was carried out: 40 mg of BSA was mixed with 31 μL of MGO in a 0.1 M phosphate buffer (pH 7.4) to obtain the control solution with 1 mg/mL BSA and 5 mM MGO. Two millilitres of the control solution were incubated at 37 °C for 6 days with or without 1 mL of the bean extracts in phosphate buffer. The percent inhibition was calculated based on the equation applied in the BSA-glucose assay as described above (excitation at 340 nm and emission at 420 nm).All values were expressed as mean ± SD. Data were analysed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test (Matlab version 7.6).p-coumaric acid, ferulic acid, and sinapic acid) and five flavonoids  were determined in the different rice bean samples  reported in p-coumaric acid, ferulic acid, vitexin, isovitexin, sinapic acid and quercetin. Among all of the studied rice beans, the D0000090 (843.75 ± 30.15 μg/g) variety possessed the highest TP, which was 6.9 times greater than that of the D0000318 variety (123.09 ± 10.35 μg/g). Rice bean (Vigna umbellate) and adzuki bean (Vigna angularis) are phylogenetically closely related, and have low level of divergence based on nucleotide sequences, seed proteins and isozymes [Typical phenolics that possess antioxidant activity are known to be mainly phenolic acids and flavonoids. Phenolic acids and flavonoids are major classes of phenolic compounds that widely occur in the plant kingdom . Beans a samples . Vitexinisozymes ,22. Due The TPC, as measured by the Folin-Ciocalteu method, varied among different rice beans . In the p < 0.05)  . A similin vitro, we measured the ability of the extracts to inhibit α-glucosidase at a concentration of 10 mg/mL . Similar results have been observed by Peng et al. [BSA-glucose and BSA-MGO models were used for the evaluation of the inhibitory effect on the formation of advanced glycation end products. The results are shown in g et al.  who inveIn conclusion, rice beans have been shown to possess significant differences in phytochemical content, antioxidant activities, α-glucosidase inhibition activities and advanced glycation end product formation inhibitory activities.


Background/Purpose: The effects of chemical and physical interactions in the microenvironment of solid tumors have not been fully elucidated. We hypothesized that acidosis, hypoxia, and elevated interstitial fluid pressure (eIFP) have additive effects on tumor cell biology and lead to more aggressive behavior during tumor progression. We investigated this phenomenon using three human osteosarcoma (OS) cell lines and a novel in vitro cell culture apparatus.Materials and Methods: U2OS, SaOS, and MG63 cell lines were cultured in media adjusted to various pH levels, oxygen tension , and hydrostatic gage pressure (0 or 50 mmHg). Growth rate, apoptosis, cell cycle parameters, and expression of mRNA for proteins associated with invasiveness and tumor microenvironment  were analyzed. Levels of CA IX, HIF-1α, and MMP-9 were measured using immunofluorescence. The effect of pH on invasiveness was evaluated in a Matrigel chamber assay.Results: Within the acidic–hypoxic–pressurized conditions that simulate the microenvironment at a tumor’s center, invasive genes were upregulated, but the cell cycle was downregulated. The combined influence of acidosis, hypoxia, and IFP promoted invasiveness and angiogenesis to a greater extent than did pH, pO2, or eIFP individually. Significant cell death after brief exposure to acidic conditions occurred in each cell line during acclimation to acidic media, while prolonged exposure to acidic media resulted in reduced cell death. Furthermore, 48-h exposure to acidic conditions promoted tumor invasiveness in the Matrigel assay.Conclusion: Our findings demonstrate that tumor microenvironmental parameters – particularly pH, pO2, and eIFP – additively influence tumor proliferation, invasion, metabolism, and viability to enhance cell survival and must be controlled in OS research. Extracellular pH (pHe) and pO2 measured in malignant musculoskeletal tumors are lower than in benign tumors and normal tissues  that had the most profound effect on the growth of human OS cell lines and their expression of the proteins that contribute to metastatic potential. Further, because OS is a highly invasive cancer, we analyzed its invasiveness in vitro under acidic and hypoxic conditions. Ostensibly, control of the tumor microenvironment and the consequent altered expression of relevant proteins may allow suppression of aggressive tumor growth and the metastatic potential characteristic of high-grade malignant tumors.While acidosis, hypoxia, and IFP frequently have been examined individually, we hypothesize that these microenvironmental factors exert additive effects on tumor cell biology and may lead to more aggressive or unexpectedly variable behavior during tumor progression and metastasis. Using a novel We used three human OS cell lines  and, given the propensity for OS to metastasize to lung, we also included one lung carcinoma cell line (H1299) . U2OS and SaOS2 lines were maintained in McCoy’s 5A media supplemented with 15% fetal calf serum (FCS); MG63 was grown in MEM media with 10% FCS; and H1299 was maintained in RPMI1640 media with 10% FCS.® culture cassette system , to which 0 or 50 mmHg hydrostatic pressure (gage) was applied in a manner that we have described previously  was selected as the elevated hydrostatic pressure, corresponding to a level commonly seen in human and canine OS in vivo and consistent with our previous studies  or normoxic  conditions. Both incubators were saturated to 100% humidity and temperature was maintained at 37°C. Cell culture media was prepared using two buffer systems, a 20 mM piperazine-N,N′-bis (2-ethanesulfonic acid) buffer to formulate the pH 6.5 and 6.8 media, and standard bicarbonate buffer to formulate the pH 7.4 and 7.1 media. pH was adjusted using 0.1 M HCl.These experiments were performed using the OptiCelleviously . Briefly studies , 16, 17.5 cells/cassette; separate cassettes were provided for each set of conditions. The experimental duration for the cell cycle and cell proliferation studies was 96 h total, comprising a 24-h, post-inoculation cell attachment phase, followed by 72 h of pressurization. The control conditions were pH 7.4, normoxic pO2, and 0 mmHg. To evaluate the long-term effects of acidic conditions on all cell lines, the cultures were acclimatized, or preconditioned, for 2 weeks in 75 cm2 flasks at pH 6.5, 6.8, 7.1, or 7.4. They were kept under normoxic pO2 levels and 0 mmHg hydrostatic pressure before transferral into the OptiCell® cassettes and initiation of hydrostatic pressurization and/or hypoxia. At selected intervals, cells within the cassette were released from their membrane using trypsin, spun down, and counted to determine total viable cell count (TVCC). TVCC measured 24 h after seeding corresponded to the “0 h” time point in all studies. Net growth was expressed using the following ratio:All cells were seeded at 1.5 × 102, and under 500 mmHg of hydrostatic pressure, were collected, washed with cold PBS, and fixed in 70% methanol at 4°C. The U2OS cells, cultured for 72 h at 37°C; media pH 7.4, 7.1, 6.8, or 6.5; and 2% pO2, and under 50 mmHg of hydrostatic pressure, were similarly collected, washed, and fixed. Cells were pelleted and treated with 100 units/mL RNase, and DNA was fluorescently labeled with 0.05 mg/mL propidium iodide. The proportion of cells in the sub-G0/G1 phase and other cell cycle phases was evaluated with flow cytometry  and subsequent analysis with FlowJo software .The H1299 cells, after 72 h of growth at 37°C, media pH 6.8, and 2% pO2) or hypoxia (2% O2); and normal or elevated hydrostatic pressure (0 or 50 mmHg), was assessed via QT-PCR for the relative expression of CA IX, VEGF-A, HIF-1A, MMP-9, and TIMP-2 using the TaqMan Gene Expression Assays kit  and associated probes . The manufacturer’s protocols were followed for the standardization, validation, and analysis assays, as previously described  and treated with RNase-Free DNase I . cDNA was synthesized using a SuperScript II Wrst-Strand real-time polymerase chain reaction (RT-PCR) kit (Invitrogen). Gene expression was measured by quantitative RT-PCR  using 50 ng of rat cDNA and 2× TaqMan Universal PCR Master Mix (Applied Biosystems) with a one-step program . The hypoxanthine phosphoribosyl-transferase (HPRT) gene was used as an endogenous normalizer. Duplicate samples without cDNA (no template control) for each gene showed no contaminating DNA.4 cells. After culturing the cells at pH 7.4 for 24 h, the medium was replaced with pH 7.4, 6.8, or 6.5 media and cells were cultured in either 2% O2/98% N2 or 20% O2/80% N2 for an additional 12, 24, 36, and 48 h. The cells on the glass slides were washed with PBS and fixed with 4% paraformaldehyde.Cells were grown in the Nunc Lab-Tek II 4-well Chamber Slide System ; each well was seeded with 5 × 10An automated immunofluorescence staining protocol was performed with a Discovery XT processor . Rabbit polyclonal antibodies were used for the primary incubation. The CA IX antibody  was used at a concentration of 2.5 μg/mL, and the HIF-1α antibody  was used at 12 μg/mL. Before the primary antibody incubation, the glass slides were blocked for 30 min in 10% normal goat serum, 2% BSA in PBS. Incubation with the primary antibody was 3 h, followed by 60 min incubation with biotinylated goat anti-rabbit IgG  diluted 1:200. Normal rabbit IgG (5 μg/mL) was used as an appropriate isotype-negative control. The detection was performed with Streptavidin-HRP D , followed by incubation with either Tyramide-Alexa Fluor 488  for CA IX or with Tyramide-Alexa Fluor 568  for HIF-1α.Cells were visualized using fluorescence microscopy (ZEISS Axioplan2 Imaging). ZEISS AxioVision (ver. 4.6.3.SP1) and Adobe Photoshop  were used to analyze the quantity of each protein level. Fluorescence photographs were taken with light exposure times of 400 ms for CA IX and 500 ms for HIF-1α; two photographs were taken in each group. Fluorescence was quantified with Photoshop, using a previously published method of evaluating luminosity . Histogr2, 0 mmHg), 5 × 104 cells of U2OS, 15 × 104 cells of MG63, or 5 × 104 cells of SaOS2 were added to the invasion chamber and cultured for 24 h in pH 7.4 media under 2 or 20% pO2. FCS was used as the chemoattractant; cells incubated without a chemoattractant served as controls. After 24 h, non-invasive cells were removed from the upper surface of the membrane with a cotton swab. The invasive cells on the lower surface of the membrane were fixed with 4% methanol, stained with toluidine blue, and counted in four separate areas with an inverted microscope . An “invasion index” was computed using the manufacturer’s analysis protocol, in which the invasion index was defined as the quotient of the percent cell invasion in a test chamber divided by the percent cell invasion in a control chamber.An invasion assay was performed on the OS cell lines using the Biocoat Matrigel Invasion Chamber (Becton Dickinson). After culturing the OS cells in pH 7.4 or 6.8 media for 48 h (20% pOt test was used to assess the significance of any differences between the results of each condition relative to control. Each data point shown in the figures is the mean ± standard error (n = 3 cassettes/condition), and significance is defined as P < 0.05.Student’s 2, and hydrostatic pressure levels after 2 weeks of preconditioning at acidic pH levels or normal pH  was used to calculate net growth of U2OS cells at various pH, pOH Figure A. The peH Figure A. PrevioH Figure ; howeverH Figure A. In addH Figure B; the sl2, and 0 mmHg hydrostatic pressure; 20% pO2, and 50 mmHg hydrostatic pressure; 2% pO2 and 0 mmHg hydrostatic pressure; and 2% pO2 and 50 mmHg hydrostatic pressure. Comparison with cells grown under pH 7.4 and 20% pO2 (control condition), suggests that the TVCC ratio of all tumor cell lines is reduced in acidic media. At pH 6.8, the growth of all tumor cells is not promoted by hypoxia or elevated hydrostatic pressure. For all tumor cell lines, no significant difference was observed in cell cycle and apoptosis rates of cells preconditioned in acidic media and subsequently grown in acidic media, whether under elevated hydrostatic pressure, hypoxia, or a combination of hypoxia and hydrostatic pressure (data not shown). On the other hand, acidosis was associated with changes in cell cycle . The combination of acidic pH, hypoxia, and high hydrostatic pressure elicited VEGF-A levels that were 3.3-fold higher than the neutral conditions in MG63 cells and 4.1-fold higher than the control conditions in H1299 cells. These findings suggest that VEGF-A expression is additively influenced by combinations of altered microenvironmental factors.Expression of 2), this degradation is inhibited, leading to increased HIF-1A  was 5.57 in MG63 cells, 0.36 in SaOS2, 71.9 in U2OS, and 2.74 in H1299 cells. MMP-9 was more highly expressed in MG63 and U2OS cells than in SaOS2 and H1299 cells. In MG63 and SaOS2, MMP-9 expression was higher in acidic conditions than in control conditions , but in the other cell lines, acidic growth conditions didn’t increase the expression of TIMP-2. Elevated hydrostatic pressure promoted greater expression of MMP-9 in SaOS2 and H1299 cells. For MMP-9 and TIMP-2, acidic media was the most influential factor for two (MG63 and SaOS2) of three OS cell lines. Elevated hydrostatic pressure and acidic media each enhanced mRNA expression of MMP-9 in tumor cells.In Figures CA IX and HIF-1A mRNA expression was affected more by acidic pH and hypoxia than by elevated IFP. The effects of pH and hypoxia on CA IX and HIF-1α protein level expression and their relationship to tumor invasiveness were also studied using immunofluorescence with in vitro cell culture. The immunofluorescence images of colocalized CA IX and HIF-1α in U2OS are presented in Figure CA IX mRNA expression in the quantitative RT-PCR analysis . The invasion index of SaOS2 cells was not upregulated by either acidic or hypoxic conditions.The invasion indices for MG63, SaOS2, and U2OS cells are shown in Table In vitro tumor cell culture conditions typically lack the elevated pressure, hypoxia, and acidosis frequently observed in solid tumors in vivo  precludes the examination of any factors which could contribute further additive effects to tumor cell growth and metastatic potential; in the case of OS, the influences of MMP/TIMP interactions and ECM-associated growth factors on invasive potential have been examined but have not yet been fully elucidated (43A number of factors limit the extrapolation of our results to  regions . This gr regions . Third, 63 cells , 42. Finucidated , 43–47. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus , the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus.We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont  Rhodococcus comprises metabolically diverse nocardioform high-G+C-content actinomycetes that have been isolated from a wide variety of sources . This neglected tropical disease is associated with poverty, lacks both a vaccine and an effective drug treatment for its chronic phase, and causes >12,000 mortalities annually through damage to the heart and central nervous system of humans . A single colony of Rc. rhodnii was grown in tryptic soy broth (TSB) at 28°C, with 250 rpm shaking, for 4 days. Total genomic DNA was isolated using the Fast DNA spin kit for soil  and quantified using a Quant-iT BR DNA assay (Invitrogen). Five hundred nanograms of genomic DNA was fragmented by nebulization and used to make a 454 Lib-L sequencing library, which was shotgun sequenced using a 454/Roche GS-FLX Titanium platform . This generated 601,588 reads, with an average length of 437 bp, and total of 262,813,142 bp. Reads were assembled into 140 contigs using the GS de novo assembler (version 2.3) with default parameters, aligning 99.45% of reads, with a mean read depth of 56.0. The largest contig size is 223,927 nucleotides (nt). Annotation was performed using xBASE2 (1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxal (B6), biotin (B7), tetrahydrofolate (B9), and cobalamin (B12).APMY00000000. The version described in this paper is the first version, accession no. APMY01000000.This Whole-Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from  Cancer is a leading cause of death in industrialized countries Cacospongia scalaris, exerts an anti-inflammatory effect by inhibiting PLA2 activity in vitro studies reported that scalaradial inhibited EGF-stimulated Akt Fasciospongia cavernosa, is known to inhibit NF-κB activation, leading to an anti-inflammatory effect In the last decades, marine natural products, and particularly sesterterpenoids, have played an important role in the discovery and development of new drugs for their wide variety of chemical structures and biological activities In the present study, we examined the ability of scalaradial and cacospongionolide to inhibit proliferation and induce apoptosis in several human carcinoma cell lines.Cacospongia scalaris and Fasciospongia cavernosa, respectively, as previously reported The natural compounds scalaradial and cacospongionolide were extracted from the sponges We investigated the effect of SC (10 µg/ml) or CSP (10 µg/ml) on the morphologic changes visualised by phase-contrast microscopy of T47D human breast cancer cells after 24 h. The untreated cells showed a normal morphologic aspect. Cells treated with SC or CSP were suggestive of apoptosis by blebs recognition. Dauno exhibited the same effect .n = 3) as compared to control, the untreated cells (3.12±0.25%). Dauno similarly induced the same effect (44.6±1.25%)  .m) precedes apoptosis and may represent an early signalling event m. As shown in via caspase  and p65 . Dauno (100 µM) exhibited the same effects   or SC (10 µg/ml) for 24 h reduced nuclear translocation of both p50  .m dissipation and DNA fragmentation, which were confirmed by optical microscopy, comet assay and FACS. Progresses in cancer biology and genetics have led to the concept that deregulation of apoptosis-related proteins or genes bears prominent effect on the malignant phenotype via p53. These results suggest that activated caspases are key molecules to determine the effect of SC and CSP. Thus, the significant reduction of several anti-apoptotic proteins expression suggests promising anticancer properties for both compounds. It is well known that many pro- and anti-apoptotic proteins are regulated at transcriptional level by NF-κB Cancer is a malignant disease resulting from several causes No specific permits were required for the described field studies. No specific permissions were required for collecting these sponges because the location they were taken from is not privately-owned or protected in any way. In addition, this study does not involve endangered or protected species.Cacospongia scalaris, while Cacospongionolide (CSP) was isolated from the sponge Fasciospongia cavernosa. Both sponges were collected in Rovijni (Croatia) in summer 2007, and kept in freezer at -20°C until analysis. The isolation of SC and CSP was performed as previously described Scalaradial (SC) was isolated from the sponge 2. HCT116 human colon carcinoma cells were cultured in McCoy’s 5A medium containing 10% FCS, 100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate . All media and supplements for cell culture were purchased from Gibco-Invitrogen . Cells (3×105) were seeded in 24-well plates and grown for 24 h before treatment with scalaradial , cacospongionolide , daunorubicin  or cisPlatinum  for the indicated period. Cell lines were all from ATCC catalogue. In some experiments, cells were incubated with the pan-caspase inhibitor Z-VAD-fmk (50 µM) or the p53 inhibitor pifthrin-alpha (30 µM) in the presence of SC or CSP.T47D (breast carcinoma), A431 (epidermoid carcinoma), HeLa (cervix adenocarcinoma) human cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 1 mM pyruvate and 10 mM Hepes at 37°C under 5% COThe cells were plated in 96 culture wells at a density of 250.000 cells/ml and allowed to adhere for 2 h. Thereafter the medium was replaced with fresh medium and cells were incubated with SC, CSP or Dauno alone or in association. After 24 h, cell viability was determined by using 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion assay 6 cells/ml) were plated in 35×10 mm diameter culture dishes and incubated with SC (10 µg/ml), CSP (10 µg/ml) or Dauno for 24 h. Thereafter, the cells were washed three times with PBS, fixed with 3% formaldehyde in PBS, and then visualised by phase-contrast microscopy .T47D cells (1×10The comet assay was performed according to a standard protocol 6) cells were seeded on 10 cm diameter Petri dishes and allowed to adhere for 2 h. Thereafter the medium was replaced with fresh medium and the cells were incubated as described above for 24 h. For analysis of genomic DNA, cells were gently scraped off and collected together with non-attached cells in the supernatant. Cells were resuspended in 250 µl of Tris-Borate EDTA (TBE) buffer containing 0.25% NP-40 and 100 µg/ml RNAse A. After incubation at 37°C for 30 min., extracts were treated with 1mg/ml proteinase K for additional 30 min. at 37°C. Then equal amounts (10 µl) of the extracts were loaded on a 1% agarose gel containing (1 µg/ml) ethidium bromide and run for 1h in 1×TBE at 90 V. The gel captions were obtained with Polaroid T667.T47D (2×106) were seeded on 10 cm diameter culture dishes and incubated with CSP, SC or Dauno for 24 h. Thereafter, the cells were scraped off the culture plates, centrifuged at 180×g for 10 min, resuspended in 250 µl of TE-buffer , and incubated with an additional volume of lysis buffer  for 30 min at 4°C. After lysis, the intact chromatin (pellet) was separated from DNA fragments (supernatant) by centrifugation for 15 min at 13 000 rpm. Pellet was resuspended in 500 µl of TE-buffer and samples were precipitated by adding 500 µl of 25% trichloroacetic acid at 4°C. Samples were pelleted at 3000×g for 10 min and the supernatant was removed. After addition of 300 µl of 5% trichloroacetic acid, samples were boiled for 15 min. DNA contents were quantified by using the diphenylamine (DPA) reagent . The percentage of fragmented DNA was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.DNA fragmentation was performed essentially as previously described by Messmer and Brune 6(3), 40 nM) for quantification of the mitochondrial transmembrane potential (ΔΨm) and propidium iodide  (both from Molecular probes) for determination of cell viability The following fluorochromes were employed to determine apoptosis-associated changes by cytofluorometry: 3,3′-dihexyloxacarbocyanine iodide (DiOCvs. untreated cells).The expression profile of apoptosis-related proteins was detected and analyzed using a human apoptosis array kit (ARY009), according with the manufacturer’s instructions (R&D Systems). Briefly, the membrane containing immobilized apoptosis-related antibodies was blocked with bovine serum albumin for 1 h on a rocking platform at room temperature. The membrane was then incubated with lysates of untreated or treated HeLa cells  along with Detection Antibody Cocktail overnight at 2°C to 8°C on a rocking platform. The membrane was incubated with streptavidin-horseradish peroxidase conjugate followed by chemiluminescent detection reagent. The membrane was scanned and pixel density was presented by quantifying the mean spot densities from two separate experiments. For quantification, the spot volume was determined, corrected for background and expressed as fold change  and Bonferroni-corrected P-value for multiple comparison test. The level of statistically significant difference was defined as p<0.05.Results are expressed as the means ± SEM of


Mycobacterium tuberculosis presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistance within M. tuberculosis populations with excellent accuracy versus the agar proportion method. We designed dual TaqMan-MGB probes to detect wild-type and mutant sequences of katG (315), rpoB (531), gyrA  and rrs (1401), genes that associate with resistance to isoniazid, rifampin, fluoroquinolone, and aminoglycoside respectively. We generated heteroresistant mixtures of susceptible and extensively drug resistant Tb, followed by DNA extraction and digital PCR. Digital PCR yielded a close approximation to agar proportion's percentages of resistant colonies, and yielded 100% concordance with agar proportion's susceptible/resistant results. Indeed, the digital PCR method was able to identify mutant sequence in mixtures containing as little as 1000∶1 susceptible:resistant Tb. By contrast, real-time PCR or PCR followed by Sanger sequencing were less sensitive and had little resolution to detect heteroresistance, requiring fully 1∶1 or 10∶1 susceptible:resistant ratios in order to detect resistance. Our assay can also work in sputum so long as sufficient quantities of Tb are present (>1000 cfu/ml). This work demonstrates the utility of digital PCR to detect and quantify heteroresistance in drug resistant Tb, which may be useful to inform treatment decisions faster than agar proportion.Drug resistance in  Multidrug-resistant tuberculosis (MDR-TB), defined as resistance of >1% of colonies to isoniazid (INH) and rifampin (RIF), was found in 3.3% of new cases and 21% of recurrent TB cases globally M. tuberculosis and has been reported for isoniazid, rifampin, ethambutol, streptomycin, and fluoroquinolones Heteroresistance allows subpopulations of bacteria to grow despite the presence of antibiotics and is thus a precursor to full resistance rpoB mutant DNA is required to be detected by the Xpert MTB/RIF assay gyrA gene, but again required the presence of 5–10% of mutant DNA to be detected in a mixture with wild type DNA Most of the latter PCR-based methods are laborious and technically difficult. Moreover, detection of low amounts of mutant template is challenging with PCR since the abundant (in this case wild-type) sequence may preferentially amplify. This possibility urges caution given the proliferation of PCR-based methods to detect MDR TB in the field. For example, it has been reported that between 65% and 100% of In 1999 the term digital PCR was used to describe dilution of DNA to single copies to improve the sensitivity of PCR to detect rare cancer-associated mutations amidst an excess of wild-type sequence M. tuberculosis H37Rv (ATCC 27294) and a clinical extensively drug resistant (XDR) TB isolate  from the Mycobacteriology Service Unit, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. All work was approved by the University of Virginia Institutional Biosafety Committee and Human Investigation Committees. Tb isolates were cultured on Lowenstein-Jensen medium at 37°C for two weeks. Cell suspensions were prepared in Middlebrook 7H9 (M7H9) broth supplemented with Middlebrook OADC enrichment  and adjusted to 0.5 McFarland.Mycobacterial strains used in this study included The 0.5 McFarland XDR-TB was diluted 10-fold serially in M7H9 plus OADC. One ml of each XDR-TB suspension  was mixed with 1 ml of 0.5 McFarland H37Rv to construct mixtures of 1∶1, 10∶1, 100∶1, and 1000∶1 respectively.DNA was isolated from each TB mixture. Briefly, 1 ml of 0.5 McFarland H37Rv, XDR-TB and each H37Rv:XDR-TB mixtures were transferred to 2 ml screw cap tube, centrifuged at 21,000×g for 10 min, and the pellets were re-suspended in 1 ml Tris-EDTA (TE) buffer, boiled at 100°C for 30 min, centrifuged at 21,000×g for 3 min, and supernatants were stored at −20°C as DNA template.katG, rpoB, gyrA, and rrs were sourced based on sequences of H37Rv and the XDR-TB isolate. The rpoB primers and the mutant probe were those reported by Espasa et al katG were those of Yesilkaya et al gyrA, rrs, and the rpoB wild-type probe were designed for this work using Primer Express3 . All sequences are shown in Primers and probes for katG (0.16 µl), 0.04 µl of 50 µM of each wild-type and mutant probe, 2.72 µl nuclease-free water except for katG (2.6 µl) and µl of DNA template. Real-time PCR of each gene was performed on a ViiA7  with initial denaturation at 95°C for 10 min, followed by 45 cycles denaturation at 95°C for 15 sec, and annealing and extension at 63°C for 1 min.PCR mixtures (10 µl) consisted of 5 µl of 2×Quantitect Multiplex NoROX PCR master mix , 0.1 µl of 50 µM of each forward and reverse primer except for rpoB in 10 replicates in order to calculate the percentage of positive detections at each dilution. The 1/10000 dilution was chosen and used to perform digital PCR for each gene. Digital PCR mixtures (10 µl) consisted of 5 µl of 2×Quantitect Multiplex NoROX PCR master mix , 0.1 µl of 50 µM of each forward and reverse primer except for katG (0.16 µl), 0.04 µl of 50 µM wild type probe, 0.04 µl of 50 µM mutant probe, 2.72 µl nuclease-free water except for katG (2.6 µl) and 2 µl of diluted DNA template. Digital PCR was performed in 384 well formats on a ViiA7  with initial denaturation at 95°C for 10 min, followed by 45 cycles  denaturation at 95°C for 15 sec, annealing and extension at 63°C for 1 min.DNA from each H37Rv:XDR-TB mixture was diluted 10 fold serially in nuclease free water and subjected to real-time PCR for rpoB in 10 replicates in order to calculate the percentage of positive detections at each dilution. The dilution that yielded approximately 50% PCR positive was chosen and for digital PCR.The 1∶1, 10∶1, 100∶1, and 1000∶1 H37Rv:XDR-TB mixtures were diluted 10 fold serially and 1 ml was spiked into 1 ml aliquots of a common TB-negative sputum sample, vortexed, liquefied by adding 2 ml of 100 µg/ml dithiothreitol (DTT), heated at 37°C for 20 min, followed by DNA extraction using Sputum DNA Isolation Kit  per the manufacturer's instructions. DNA from was then diluted 10 fold serially in nuclease free water and subjected to real-time PCR for katG, rpoB, gyrA, and rrs loci were amplified using the primers of Campbell et al ® PCR Purification Kit . Purified PCR products were measured spectrophotometrically, diluted with nuclease free water, mixed with primers then submitted to GeneWiz  for DNA sequencing and uploaded to GenBank (KC344734-KC344741).The Drugs used were isoniazid (INH), rifampin (RIF), ofloxacin , amikacin . INH, AMK were dissolved in sterile distilled water. RIF was dissolved in dimethyl sulfoxide. OFX was dissolved in 0.1 N NaOH. All stock solutions were stored in single use aliquots at −80°C.−2 and 10−3 were spread onto M7H10 with and without drug and incubated at 37°C. Critical concentrations endorsed by CLSI were used The drug susceptibility of H37Rv, XDR-TB and the population of H37Rv and XDR-TB in each mixture was tested in Middlebrook 7H10 agar (Difco) by standard procedure with slight modifications rpoB, Ser315Thr of katG, A1401G of rrs, and Asp94Gly and Ser95Thr mutations of gyrA gene, the most common mutations reported for rifampin, isoniazid, injectables, and quinolones, respectively gyrA wild-type probe showed high specificity for its target sequence, while gyrA mutant, rpoB mutant, both katG probes, and both rrs probes exhibited slight cross reactivity which was manageable with threshold adjustment. The rpoB wild-type probe showed substantial cross reactivity to mutant target  and mutant (0∶1) controls, were amplified by real-time PCR for each gene individually with primers and both probes, using the thresholds of The DNA of each H37Rv:XDR-TB mixture was amplified with locus-specific primers and submitted for sequencing. Traces are shown in rpoB primers and probes. The starting number of cells and input DNA are shown in rpoB and rrs) even at a 1000∶1 mixture of H37Rv:XDR TB. By contrast, real-time PCR or PCR with Sanger sequencing yielded only binary results (‘Resistant’ vs. ‘Susceptible’), and only at lower sensitivities.The DNA of each H37Rv:XDR-TB mixture was then diluted 10 fold serially and subjected to real-time PCR using the To mimic clinical specimens, the H37Rv:XDR-TB mixtures and their dilutions were spiked into sputum. Sputum underwent DNA extraction and this was diluted 10 fold serially to determine the appropriate dilution for digital PCR (approximately 1000 cfu/ml). Digital PCR was performed, and these results yielded a reasonable representation of the H37Rv:XDR-TB mixture, with an average of 74.7±1.7,22.8±2.1, 2.1±1.5, and 0.5±0.3% mutant sequence in spiked H37Rv:XDR-TB mixtures of 1∶1, 10∶1, 100∶1, and 1000∶1 respectively .This work demonstrates the principal of digital PCR for both detection and quantitation of heteroresistance in drug resistant tuberculosis. This method offered an extremely accurate representation of the percentage of resistant colonies that were found by agar proportion in a much shorter timeframe (1 day versus 21), and yielded sensitivity down to as little as 0.1% mutant DNA in the mixture. This speed alone may yield clinical value, particularly as there are instances when a Tb isolate does not grow well during susceptibility testing.The work urges caution with interpretation of many PCR-based genotypic resistance tests, as is used in line probe, Xpert MTB/RIF, and sequencing methodologies. While these methods offer significant advantages in terms of speed and simplicity of interpretation, we found that qPCR-based probe methods (on DNA from an entire Tb isolate) could detect resistance only when present at a high amount (approximately a 10% mixture). Sanger sequencing was only capable of detecting resistance at a 50/50 mixture (28–60% resistant colonies). Additionally, we examined a laboratory-developed rapid phenotypic DST assay that utilizes qPCR of mycobacteriophage 3 cfu Tb/ml sputum, rpoB is outside the variable region used for detecting other Mycobacterium species Our primary purpose of this work was to detect heteroresistance in isolates; however the digital PCR method also worked directly on sputum with sufficient Tb quantity  at various H37Rv:XDR-TB mixtures. qPCR thresholds were set per (TIF)Click here for additional data file.Table S1Starting number and amount of cells in each PCR well for each diluted DNA template.(DOC)Click here for additional data file.Table S2Specificity of each probe versus non-tuberculous mycobacteria (NTM).(DOCX)Click here for additional data file.


AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵ As a result, the two proline synthetases, P5CS and OAT, and the proline catabolic enzyme, ProDH, are mainly involved in the net proline accumulation in plants.Water deficit (WD) or drought obviously limits plant growth and productivity, and plants possess a range of morphological, physiological and biochemical adaptive mechanisms in response to WD Arbuscular mycorrhizal fungi (AMF) are able to form symbiotic associations with more than 80% of land plants for better uptake of nutrient and water Lactuca sativaMacadamia tetraphyllaOryza sativaPistacia veraCitrus tangerinaErythrina variegataKnautia arvensisPoncirus trifoliataGlycine max induced higher proline accumulation in roots but lower in shoots under WD In general, proline accumulation positively correlates with drought tolerance of AMF-colonized plants. Compared with the non-mycorrhizal plants, proline accumulation in the AMF-colonized plants exposed to WD was higher in Poncirus trifoliata L. Raf.), a citrus relative, is relatively sensitive to soil water deficit and also is highly dependent on mycorrhizal symbiosis due to less root hairs. The objective of the present work was therefore to determine the relationships between proline accumulation and activity of key proline metabolic enzymes  in trifoliate orange under AMF colonization and/or soil water deficit.Trifoliata orange (Funneliformis mosseae (syn. Glomus mosseae) (Nicol. & Gerd.) Schüßler & Walker (BGC XZ02A) has been used since trifoliate orange has higher drought tolerance under the inoculation with this native strain than other Glomus species or strains Incarvillea younghusbandii in Dangxiong, China. The AM inoculum was propagated through the identified spores with white clover (Trifolium repens) for 16 weeks and was a mixture of AM-infected roots, spores (∼23 spores/g), hyphae and sand.An AM fungus strain, 2/s photosynthetic photon flux density, 25/19°C (day/night), and 70–95% relative air humidity in a plastic greenhouse locating in the university campus.Seeds of trifoliate orange were surface sterilized with 70% ethanol for 15 min and rinsed thoroughly with distilled water. On March 15, 2012, five sterilized seeds were sown in a 18 × 16.5×13 cm (top diameter × bottom diameter × height) plastic pot filled with 2.3 kg autoclaved  soils . Soils at 0–20 cm depth were collected from a campus Citrus Orchard of Yangtze University, Jingzhou, China. This soil contained organic matter 9.8 g/kg, available N 114.2 mg/kg, Olsen-P 15.7 mg/kg, and available K 20.8 mg/kg. Before seed sowing, the AMF inoculation pots were received 40 g AM inocula at 5 cm soil depth, while the no-AMF pots were received both 40 g autoclaved  inocula and 2 ml inoculum filtrate (25 µm filter) for other microbial communities. After 40 days of sowing, three seedlings per pot were grown under 338–982 µmol/mF. mosseae) and two water treatments . The four treatments were coded as AM−/WW (no-AM and WW), AM−/WD (no-AM and WD), AM+/WW (AM and WW), and AM+/WD (AM and WD), respectively. Each treatment had three replicates, and each replicate (pot) contained three seedlings. A total of 36 seedlings (4 treatments × 9 seedlings/treatment) were used at harvest in the study. After 87 days of acclimation for mycorrhizal formation, the WW  and the WD  polyvinylpolypyrrolidone, and then centrifuged at 20,000 g for 20 min at 4°C. One ml supernatant was mixed with 3 ml 50 mM Tric-HCl containing 20 mM MgCl2, 10 mM ATP, 100 mM hydroxylamine-HCl and 50 mM glutamine. After 15 min at 37°C, the reaction was stopped by the addition of 3 ml 5 M HCl containing 5% FeCl3 and 12% trichloroacetic acid. The mixture was centrifuged at 20,000 g for 10 min at 4°C, and the absorbance of the supernatant was determined at 535 nm, with the no-ATP reactive solution as the control. One unit of P5CS was expressed as the enzyme amount of 1.0 μmol glutamate during 1 min (U/g DW). The weighted plant total activity of P5CS was calculated as (leaf activity of P5CS × leaf biomass weight + root activity of P5CS × root biomass weight)/(leaf biomass weight + root biomass weight).Activity of leaf or root P5CS was determined according to the method of Hayzer and Leisinger 1-pyrroline-5-carboxylate during 1 min (U/g DW). The weighted plant total activity of OAT was calculated as (leaf activity of OAT × leaf biomass weight + root activity of OAT × root biomass weight)/(leaf biomass weight + root biomass weight).Determination of leaf or root OAT activity was accorded to Kim et al. g for 15 min at 4°C, 10 µl TritonX-100 was added, placed for 30 min at 4°C, and centrifuged at 20,000 g for 20 min at 4°C. A total of 2.5 ml solution consisted of 0.5 ml supernatant, 1.6 ml bicarbonate buffer (pH 10.3), 0.2 ml 0.1 mM proline, and 0.2 ml 0.9 mM 2, 6-dichlorophenolindophenol. After the addition of 0.2 ml 9 mg/ml phenazine methosulfate, the absorbance of the solution increased 0.01 at 600 nm during 1 min was defined as one activity unit of ProDH (U/g DW). The weighted plant total activity of ProDH was calculated as (leaf activity of ProDH × leaf biomass weight + root activity of ProDH × root biomass weight)/(leaf biomass weight + root biomass weight).Activity of leaf or root ProDH was assayed according to Zhao et al. n = 3) were subjected to two-way ANOVA and Duncan’s multiple range tests were used to compare significant differences among treatments at the 5% level. All statistical analyses were performed with the SAS v8.1 software .Data  than under WD (52.20±4.00%). No root mycorrhizal colonization was observed in the no-AMF inoculated seedlings, regardless of the soil water status.The water deficit generally significantly decreased plant height, stem diameter, and leaf number, irrespective of AM or no-AM treatment . The AM +/WW > AM−/WW > AM+/WD > AM−/WD (Leaf relative water content (LRWC) ranged 73–80% under WW and 65–69% under WD , and sig> AM−/WD ; Table 2+/WW > AM+/WD ≈ AM−/WW > AM−/WD  under WW or 1.02–1.53 mg/g DW in leaves, 0.44–0.84 mg/g DW in roots, and 0.78–1.26 mg/g DW in total plant under WD    , signifi P<0.01) , but did P<0.01) .1-pyrroline-5-carboxylate dehydrogenase In the present study, WD induced significantly higher total plant (leaf + root) proline accumulation in trifoliate orange seedlings , in compF. mosseae colonization significantly decreased both concentrations and contents of leaf, root, and total plant proline in trifoliate orange seedlings, regardless of water status  and a mixture of eight AMF species Under WD proline accumulation was either significantly higher r status . SimilarP5CS transcript expression was in F. mosseae-colonized soybean and lettuce plants than in no-AMF colonized plants under WD. Proline biosynthesis is usually controlled by P5CS1 and P5CS2 gene in plants, and the P5CS1 can be regulated by abiotic stresses, leading to an enhanced proline biosynthesis in plastids P5CS1 gene in the proline biosynthesis of AM plants growing under WD.In the present study, a significantly lower P5CS and OAT activity of leaf and total plant was found in the AM than in the no-AM trifoliate orange seedlings under both WW and WD . The mycProDH gene is rarely expressed and the ProDH protein is more stable than the P5CS protein ProDH.Another factor contributing to proline accumulation under WD is the catabolism of proline F. mosseae increased the plant growth performance and biomass production but decreased tissue proline accumulation under either WW or WD. The total plant proline significantly positively correlated with plant P5CS activity, negatively with ProDH activity, but not with OAT activity. On the other hand, the AM plants may suffer less from WD due to better leaf water status and root volume, thereby showing lower proline accumulation. In addition, the lower proline accumulation in the AM plants may derive from the integration of the inhibition of glutamate synthetic pathway of proline with an enhancement of proline degradation, no matter whether plants grow under WD or not.In short, inoculation with


The amygdala is a critical brain region for auditory fear conditioning, which is a stressful condition for experimental rats. Adult neurogenesis in the dentate gyrus (DG) of the hippocampus, known to be sensitive to behavioral stress and treatment of the antidepressant fluoxetine (FLX), is involved in the formation of hippocampus-dependent memories. Here, we investigated whether neurogenesis also occurs in the amygdala and contributes to auditory fear memory. In rats showing persistent auditory fear memory following fear conditioning, we found that the survival of new-born cells and the number of new-born cells that differentiated into mature neurons labeled by BrdU and NeuN decreased in the amygdala, but the number of cells that developed into astrocytes labeled by BrdU and GFAP increased. Chronic pretreatment with FLX partially rescued the reduction in neurogenesis in the amygdala and slightly suppressed the maintenance of the long-lasting auditory fear memory 30 days after the fear conditioning. The present results suggest that adult neurogenesis in the amygdala is sensitive to antidepressant treatment and may weaken long-lasting auditory fear memory. Fear is an emotion conserved in the animal kingdom as an instinctual response to potential danger, and is critical for adaptive functions of the brain. However, persistent fear can lead to maladaption and even stress-related disorders such as phobias and post-traumatic stress disorder (PTSD) Adult neurogenesis has been clearly demonstrated in the subgranular zone (SGZ) of hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricles. Environmental factors including environmental enrichment, physical exercise, aging and stress can positively or negatively modulate adult hippocampal neurogenesis The amygdala contributes to the acquisition and storage of fear memory ad libitum access to water and food (12 h light/dark cycle) under controlled (22±2°C) conditions. All experiments were carried out between 09∶00 and 17∶00. The animal care and the experimental protocol were approved by the Animal Ethics Committee of Central South University.Adult male Sprague–Dawley rats  weighting 200–250 g were used. Rats were group-housed with −1 · d−1; solution in normal saline, Watson pharmaceuticals (changzhou) co., LTD) for 2 w before auditory fear conditioning. Three days before anditory fear conditioning, some of the rats in each group (n = 19 to 20) were intraperitoneally injected with 200 mg · kg−1 bromodeoxyuidine  twice to label newborn cells. The administration interval was 12 hours.Animals were randomly divided into four groups: control, fear, saline fear , and FLX fear (FLX+ F) groups (n = 48 for each group). Rats in the Sal+F group received a saline gavage for 2 w before auditory fear conditioning, and rats in FLX+ F group received a FLX gavage  that was enclosed in a ventilated and sound-attenuated box (63 cm× 43 cm× 63 cm) for 2 min and recieved 20 trials of a neutral tone  co-terminating with an electrical foot shock, the inter-trial interval was 90 s. One min after the last shock, animals were returned to their home cage. Rats in the control group were processed with the same protocol except that the footshock was omitted. For maximal conditioning, the twenty tone-shock pairings were repeated on the next day. During the retrieval test, a Plexiglas chamber distinct from the conditioning chamber was used (chamber B). Rats were placed into chamber B and exposed to three tone-alone presentation  at 1 d, 7 d, 14 d, 21 d, 30 d or 60 d after training. Freezing behavior was defined as immobility except for respiratory movements and was quantified by trained observers that were blind to the experimental groups. The BrdU-labled rats were sacrificed immediately after the retrieval test to examine neurogenesis.Rats injected with BrdU were anesthetized  and fixed by perfusing with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and postfixed overnight in 4% paraformaldehyde at 4°C. After treatment with 30% sucrose, brains were cut into sections (40-µm thickness) through the entire amygydala and hippocampus  in the coronal plane. For stereological BrdU counts, 1 of every 12 sections throughout the entire amygdala and hippocampus were mounted onto slides, heated in citric acid  for 30 min at 95°C for antigen retrieval, and denatured in 2 N HCl for 30 min at 37°C. Sections were incubated for 15 min in 3% hydrogen peroxide to eliminate endogenous peroxidases, then rinsed and incubated with blocking buffer  for 1 h. Sections were then incubated with a mouse anti-BrdU antibody  overnight at 4°C, rinsed and transferred to biotinylated goat anti-mouse IgG  at room temperature for 2 h. BrdU was visualized using an avidin-biotin-horseradish peroxidase kit (Vector Laboratories) and reacted with diaminobenzidine (Sigma).For double labeling, immunofluorescence staining was used. Briefly, denatured sections were incubated overnight with rat anti-BrdU monoclonal IgG  and a mouse anti-NeuN monoclonal IgG , or a goat anti-GFAP polyclonal IgG  in PBS. The secondary antibodies were Cy3-conjugated donkey anti-rat IgG , and Alexa Fluor 488 goat anti-mouse IgG  or Alexa Fluor 488 Rabbit anti-goat IgG . Sections incubed in the secondary antibodies for 1 h and coverslipped with fluorescent mounting medium (Invitrogen). Fluorescence images were captured on a confocl microscope .The number of BrdU-positive cells was estimated using a modified stereology protocol For estimating the phenotype of newborn cells, slices were analyzed on a confocal microscope , Single-layer images were scanned, and Z-stacks of confocal images  were projected to reconstruct astrocyte structure P<0.05.Data are presented as group means ± SEM. Group difference were assessed with Student’s t-test or analysis of variance (ANOVA). The criterion for significance was set at P<0.001), 7 d , 14 d , 21 d , 30 d  and 60 d  after the fear conditioning , 7 d , 14 d , 21 d , 30 d  and 60 d  after auditory fear conditioning  . In contrast, the proportion of BrdU-postive cells expressing GFAP was higher in fear rats than in controls (P<0.05) (P<0.05); meanwhile, the proportion of BrdU-postive cells expressing GFAP was18.87±1.46% in fear rats compared with 13.05±1.95% in control animals (P<0.05). Similar results were obtained in the hippocampus in the first two weeks after fear conditioning, but the difference disappeared at later time points  . In con(P<0.05) . The same points . The respost hoc t tests showed a significant difference between Sal+F group and FLX+F group, as well as between FLX+F group and control group 1 d  = 103.283, P<0.001), 7 d  = 69.452, P<0.001), 14 d  = 34.187, P<0.001), 21 d  = 72.321, P<0.001) and 30 d  = 20.238, P<0.001) after auditory fear conditioning. The number of BrdU-positive cells in FLX treated (FLX+F) rats was significantly increased at 1 d , 7 d , 14 d , 21 d  and 30 d  time points but were still lower than naive controls  = 10.467, P<0.05), 7 d  = 11.923, P<0.01), 14 d  = 7.035, P<0.05), 21 d  = 24.059, P<0.01), 30 d  = 27.654, P<0.01) and 60 d  = 16.317, P<0.01) after auditory fear conditioning. Compared to saline control of the animals underwent fear conditioning, the proportion of BrdU-postive cells expressing NeuN was increased in FLX treated animals 1 d , 7 d , 14 d , 21 d , 30 d  and 60 d  after auditory fear conditioning  = 5.887, P<0.05), 14 d  = 7.038, P<0.05), 21 d  = 6.944, P<0.05), 30 d  = 8.965, P<0.05) and 60 d  = 6.494, P<0.05) after auditory fear conditioning. Compared with Sal+F group, the ratio of BrdU-postive cells expressing GFAP was significantly decreased in FLX+F group 7 d , 14 d , 21 d , 30 d  and 60 d  after auditory fear conditioning  and 60 d (P<0.05) after fear conditioning (As expected, we observed that FLX suppressed the maintenance of the long-term auditory fear memory. In the FLX and saline treated fear rats, auditory fear memory was similar at 1 d, 7 d, 14 d and 21 d after auditory fear conditioning (The main findings of the present study were that the persistent auditory fear memory induced by fear conditioning led to decrease the survival and an alteration in the differentiation pattern of new-born cells in the amygdala. Chronic treatment with FLX before auditory fear conditioning partially rescued the impairment of the amygdaloid neurogenesis and suppressed long-lasting auditory fear memory in rats.Fear conditioning is a widely used as a model of PTSD FLX treatment is known to increase adult neurogenesis in the rodent hippocampus Our study also showed that the number of adult-born cells in the amygdala reached to peak at 7 days after fear conditioning, and two months after the fear conditioning, only about forty percent of BrdU-positive cells expressing NeuN in the amygdala in control rats, which suggested that adult-born cells in the amygdala have longer proliferation time periods and fewer cells attain a mature phenotype compared to those in the hippocampus. These data could contribute to a new understanding for the treatment of PTSD based on the distinct characteristics of the adult neurogenesis in the amygdala and hippocampus.In the study, we used BrdU to investigate the relationship between amygdaloid neurogenesis and auditory fear memory. BrdU can incorporate DNA of dividing cells during the S-phase of the cell cycle, so not all BrdU-labeled cells are neural cells, which is the limitation of BrdU, through BrdU-labeling is the most used method for studying adult neurogenesis.The present study used auditory fear conditioning to elucidate the relationship between amygdaloid neurogenesis and auditory fear memory. The results demonstrate that auditory fear conditioning induced persistent auditory fear memory, which was associated with suppression of the survival and maturation of new-born cells in the amygdala. FLX treatment partially reversed the impairments in amygdaloid neurogenesis, leading to the suppression of long-term fear memory. Overall, our data suggest that adult neurogenesis in the amygdala may be contribute to auditory fear memory.


To this end, obese mice were induced by feeding with high fat diet  and the age-matched control mice were fed with control diet  for 6 months. Simultaneously these mice were treated with or without BL153 daily at 3 dose levels  by gavage. HFD feeding significantly increased the body weight and the liver weight. Administration of BL153 significantly reduced the liver weight but without effects on body weight. As a critical step of the development of NAFLD, hepatic fibrosis was induced in the mice fed with HFD, shown by upregulating the expression of connective tissue growth factor and transforming growth factor beta 1, which were significantly attenuated by BL153 in a dose-dependent manner. Mechanism study revealed that BL153 significantly suppressed HFD induced hepatic lipid accumulation and oxidative stress and slightly prevented liver inflammation. These results suggest that HFD induced fibrosis in the liver can be prevented partially by BL153, probably due to reduction of hepatic lipid accumulation, inflammation and oxidative stress.The present study was to investigate whether a  Obesity is becoming a health issue all over the world. It grows rapidly and always leads to severe complications such as cardiovascular disorder, diabetes, and cancer –3. LiverMagnolia officinalis is regarded as Chinese traditional medicine and used in the clinical practice for a long time to treat various disorders [ Magnolia such as honokiol, obovatol, and magnolol have been reported to have antioxidative [κB activation, activin phosphorylation, and subsequent IκBα degradation [κB is that honokiol decreases levels of NF-κB target genes including tumor necrosis factor (TNF-) α, intercellular adhesion molecule (ICAM-) 1, and plasminogen activator inhibitor (PAI-) 1. Honokiol also plays critical role in scavenging reactive oxygen species via inhibition of TNF-α mediated NADPH oxidase (NOX) pathway in hepatocytes [ Magnolia, magnolol, and obovatol also showed the antioxidative effect by attenuation of ROS generation and the subsequent reduction of NF-κB activation [β1) and its downstream profibrotic factors such as connective tissue growth factor (CTGF) through inhibition of Smad-2/3 signaling pathway [β-oxidation and lipolysis, finally reducing lipid accumulation in the tissue [isorders , 10. Sevxidative , 12 and xidative –15. Honoradation , 17. Conatocytes . In additivation , 20. Bot pathway . Furthere tissue , 23.The present study was to clarify whether BL153 has protective effects on HFD-induced liver damage and if so what is the possible mechanism. We found administration of BL153 significantly prevented chronic obesity-induced liver damage with the mechanism of reducing lipid accumulation and inhibiting inflammation and the associated oxidative stress.Magnolia extract (BL153) was prepared as our previous report [ magnolia extract have been defined in previous studies [n = 5) and fed either a control diet  or a high fat diet  with or without BL153 for six months: (1) Ctrl group: mice were fed a control diet and supplemented with 0.5% ethanol; (2) HFD group: mice were fed a HFD and supplemented with 0.5% ethanol; (3) HFD+2.5 mg/kg group: mice were fed a HFD and supplemented with BL153 at the dose of 2.5 mg/kg; (4) HFD+5 mg/kg group: mice were fed a HFD and supplemented with BL153 at the dose of 5 mg/kg; and (5) HFD+10 mg/kg group: mice were fed a HFD and supplemented with BL153 at the dose of 10 mg/kg. Selection of 5 mg/kg and 10 mg/kg for the present study was based on a previous study [s report . The maj studies –26. All us study , where t2O into final concentration of 1.0 mg/mL (high dose group), 0.5 mg/mL (middle dose group), and 0.25 mg/mL (low dose group) with final concentration of ethanol at 0.5%, respectively. Therefore, the gavage volume was 1% (mL/g) of mouse body weight . Control groups were given same volume of ddH2O with 0.5% ethanol. During the six-month feeding, body weight was measured every month, and the gavage volume was justified based on the body weight change. At the end of experiment, all mice were sacrificed and livers were collected for further analysis.For preparing BL153 gavage solution, different doses of BL153 were dissolved into 100% ethanol and then diluted with ddHThe fixed liver tissue was cut into 3 mm thickness blocks. The tissue blocks were embedded in paraffin and cut into 4 µm slices. After being deparaffinized using xylene and ethanol dilutions and rehydration, the sections were stained with hematoxylin and eosin  to examine the tissue structure, inflammatory cell infiltration, necrosis, and lipid accumulation as described previously , 28. ForCryosections from OCT-embedded tissue samples of the liver (10 mm thickness) were fixed in 10% buffered formalin for 5 min at room temperature, stained with Oil Red O for 1 h, washed with 10% isopropanol, and then counterstained with hematoxylin for 30 s. A Nikon microscope  was used to capture the Oil Red O-stained tissue sections at 40x magnification. For hepatic triglyceride levels assay, 200 mg of hepatic tissues was homogenized at 4°C in 2.0 mL diluted Standard Diluent using a Polytron tissue homogenizer. After centrifugation at 10000 ×g for 10 min at 4°C, samples were diluted by the ratio of 1 : 5 using the diluted Standard Diluent. Then, the triglyceride levels in liver tissue were measured according to the manufacturers' instructions provided in the triglyceride colorimetric assay kit .β-actin , PAI-1 , TNF-a (1 : 500), and TGF-β1 .Western blot assays were performed as described before . BrieflyP < 0.05.Data were collected from five animals for each group and presented as mean ± SD. One-way ANOVA was used to determine general difference, followed by a post hoc Tukey's test for the difference between groups using Origin 7.5 laboratory data analysis and graphing software. Statistical significance was considered as In order to identify whether BL153 can prevent obesity and the subsequent hepatic injury, HFD treatment was applied in this study to induce obesity mouse model. After 6 months of HFD feeding, the body weight was significantly increased, which indicated the establishment of the obesity mouse model  but treaβ1. Similar protective effects were observed that HFD significantly increased hepatic TGF-β1 expression, which was remarkably reduced by treatment of BL153 in a dose-dependent manner (Liver weight increase is a feature of hepatic hypertrophy which is closely associated with liver fibrosis –32. Moret manner .The above study revealed that BL153 significantly prevented HFD-induced hepatic hypertrophy and fibrosis. And lipid accumulation is the first step of NAFLD development , 36. Thuα, and PAI-1 was detected. Western blot assay revealed that HFD significantly upregulated the expression of TNF-α (α (Inflammation is the main pathological consequence of HFD-induced obesity characterized by release of inflammatory factors which contributes to insulin resistance –39. Thusof TNF-α , ICAM-1 of TNF-α , and PAIof TNF-α  in the lTNF-α (α , ICAM-1 TNF-α (α , and PAITNF-α (α , while nHFD-induced obesity generally leads to oxidative stress via release of multiple adipokines which in turn generates excessive reactive oxygen species . FurtherObesity is currently a worldwide epidemic and among the most challenging health conditions. A major metabolic consequence of obesity is insulin resistance which underlies the pathogenesis of the metabolic syndrome such as NAFLD, the hepatic manifestation of obesity and metabolic syndrome , 43. NAF magnolia extract, BL153, attenuated obesity-associated liver damage in a HFD-induced obesity mouse model. Most importantly, BL153 treatment significantly attenuated obesity caused liver pathological changes, including hepatic hypertrophy, lipid accumulation, fibrosis, inflammation, and oxidative stress.In the present study, we provide the first evidence thatMagnolia has been used as Chinese traditional medicine to treat various disorders [ Magnolia bark, reduces the number of intracellular stored lipid droplets by enhancing lipolysis and thus inhibits the formation of intracellular cholesterol esters [isorders , 10. It l esters . It is u Magnolia also showed great beneficial effect on antifibrosis. Administration of Magnolia cannot only prevent the cardiac fibrosis induced by ischemia/reperfusion but also the renal fibrosis induced by TGF-β1 [β1 expression, which were remarkably attenuated by BL153 treatment in a dose-dependent manner (Liver weight increase is a main feature of hepatic hypertrophy associated with hepatic fibrosis, which is a key step during the development of NAFLD , 49. Groy TGF-β1 , 50. In t manner . Magnolia, honokiol, plays inhibiting role on lipid accumulation-induced inflammation and oxidative stress [α, ICAM-1, and PAI-1 (As we know, inflammation and the associated oxidative stress also participate in the development of NAFLD. Moreover, other studies also mentioned that another constituent ofe stress –15, 51. nd PAI-1  as well nd PAI-1 . Adminisnd PAI-1 . BL153 and PAI-1 , which i magnolia extract BL153 can simultaneously induce beneficial effects on HFD-induced liver damage by inhibiting hepatic lipid accumulation, inflammation, oxidative stress, hypertrophy, and fibrosis.In summary, the fat liver damage is a main consequence of the development of obesity induced by HFD. The