Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries.C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates  were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17.Genetic diversity of Asian isolates of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.The findings showed a different distribution of genotypes of  Cryptococcus neoformans and C. gattii are encapsulated basidiomycetous yeasts that can cause life-threatening infections in humans. According to the current classification, C. neoformans consists of two varieties, namely variety grubii (serotype A) and variety neoformans (serotype D). C. neoformans and C. gattii differ in ecology, biochemistry, molecular characteristics, and their ability to cause disease C. neoformans is known as an opportunistic pathogen because it mainly infects immunocompromised patients, whereas C. gattii is considered a primary pathogen that infects otherwise healthy individuals C. neoformans and C. gattii differ as well. The latter species causes more frequently cryptococcomas and has a lower susceptibility to antifungal agents, resulting in prolonged treatment and a higher mortality rate when compared to C. neoformansCryptococcus neoformans continues to be the most important cause of fungal meningitis in immunocompromised patients. The global burden of cryptococcal infections among HIV-infected patients is estimated at nearly one million new cases per year C. neoformans var. grubiiC. neoformansC. neoformans/C. gattii complex, several molecular methods have been used, such as AFLP, M13-based PCR fingerprinting, Multi-locus Sequence Typing (MLST) and analysis of the intergenic spacer (IGS) ribosomal DNA sequences Most cryptococcal meningitis cases in Asia are caused by Aspergillus fumigatus and A. flavus, molecular typing using microsatellites proved to be more discriminatory than MLST In vitro antifungal susceptibility was determined for amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole. The aims of this study were: (i) to analyze the genotypic diversity as well as the distribution of C. neoformans var. grubii from different geographical regions in Asia, (ii) to relate the genetic background of the cryptococcal isolates to disease status and origin from the human body, (iii) to test the in vitro antifungal susceptiblity of the isolates against seven antifungal drugs, and (iv) to determine if differences in susceptibility correlate with the observed genotypic diversity.Microsatellite analysis is a genotyping technique that is becoming increasingly popular for molecular typing of medically important fungi Cryptococcus neoformans var. grubii isolates were obtained from the collections of the Chinese Cryptococcus Reference Centre at the Second Military Medical University, Shanghai, China (n = 115); Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (n = 79); Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India (n = 61); Sappasitthiprasong Hospital, Ubon Ratchathani, Thailand (n = 59); Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia (n = 40); Department of Microbiology, Meiji Pharmaceutical University, Tokyo, Japan (n = 28); Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand (n = 20); Department of Microbiology, Faculty of Medicine, Health Sciences Centre, Kuwait University, Jabriya, Kuwait (n = 10); Mycology Unit, Microbiology Division, Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha, Qatar (n = 5) and Department of Pathology, Faculty of Medicine, Prince of Songkla University, Hat Yai , Thailand (n = 9) (n = 58) and the Department of Microbiology, Meiji Pharmaceutical University, Tokyo, Japan (n = 9)  . Further (n = 9) . Two-hunl-Canavanine-glycine-bromothymol blue (CGB) medium was used to distinguish between C. neoformans and C. gattii isolates Cryptococcus isolates were stored in sterile 2 ml screw-capped tubes containing porous beads  at −80°C until further use.Species identification was initially performed by standard mycological methods 2Genomic DNA extraction was performed as previously described Microsatellite analysis was performed using nine microsatellite markers In vitro antifungal susceptibility testing of amphotericin B , flucytosine , fluconazole , itraconazole , posaconazole , voriconazole  and isavuconazole  was performed using the standard broth microdilution method as described in CLSI document M27-A3 Candida krusei ATCC6258 and Candida parapsilosis ATCC22019 were used as quality controls.C. neoformans. However, a MIC≤1 µg/ml was suggested as the susceptibility breakpoint for ITR, VOR, POS and ISA The resistance breakpoints of FLU and 5FC were taken from CLSI document M27-A3 The Simpson's index of diversity was calculated to assess the genotypic diversity of microsatellite genotypes among the different populations In vitro antifungal susceptibility testing results were statistically analyzed using the t-test method. P values less than 0.05 were considered significant for all statistical methods. The statistics were analyzed using StatsDirect v2.7.8 .Chi-square and Fisher exact tests were applied to examine the correlation between MCs and isolate categories, including HIV status, sample sources , and geographical origin. C. neoformans var. grubii isolates obtained from HIV-positive patients (n = 236), HIV-negative patients (n = 156), and those with an unknown HIV status (n = 34), 425 isolates (99.8%) belonged to mating-type α and serotype A (αA), and one isolate (0.2%) from a HIV-negative patient in China belonged to mating-type α and serotype AD (αAαD). All 67 environmental isolates of C. neoformans var. grubii obtained from avian droppings from Chiang Mai, Thailand (n = 58) and Tokyo, Japan (n = 9) were mating type α and serotype A  and one αAαD hybrid isolate was analyzed by microsatellite typing. Within this collection of 493 isolates, 265 different genotypes were found. These 265 genotypes were distributed over eight microsatellite complexes (MCs), which were defined as described previously n = 206, 41.8%), followed by MC2 , MC3 , MC16 , MC17 , MC1 , MC12  and MC15 . The distribution of MCs was found to differ in each country (The genetic diversity of 492 isolates of  country . MC2 was country . The relC. neoformans var. grubii was found to be most diverse (D = 1.000) in the Kuwait and Qatar populations, followed by the Japanese (D = 0.998), Indonesian (D = 0.994), Indian (D = 0.983), Chinese (D = 0.975), and Thai populations (D = 0.968) (p<0.0001) and Fisher exact tests (p<0.0001) . In contrast, MC8 was the predominant MC containing isolates from HIV-positive patients , followed by MC2 , MC3  and MC17 .The genotypic diversity of = 0.968) . The gen<0.0001) . The majp = 0.0004, Chi-square; p = 0.0001, Fisher exact test). However, no such correlation was observed within the Thai population from Chiang Mai  nor within the Japanese population from Tokyo .The distribution of environmental and clinical isolates among MCs was studied using two populations from Tokyo, Japan and Chiang Mai, Thailand. In both cases, the clinical and environmental isolates belonged to the same MCs . MC2 andC. neoformans var. grubii isolates for the seven antifungal compounds tested are listed in n = 13), Thailand (n = 4) and China (n = 1) were resistant to 5FC with MIC ≥32 µg/ml (n = 4), MC8 (n = 8) and MC17 (n = 5), and one belonged to MC2  when compared to the presence of 3.9 and 9.5% of resistant isolates in MC8 and MC3, respectively. Ten FLU-resistant isolates (2%) occurred in different countries, including China (n = 2), India (n = 1), Indonesia (n = 5), and Thailand (n = 2) and belonged to MC2 (n = 3), MC3 (n = 2), MC8 (n = 2), MC17 (n = 2) and one isolate from India (number 25_17) that could not be assigned to any MC (n = 1) (n = 2), MC8 (n = 1) and MC17 (n = 2) were resistant to both 5FC and FLU. Isolates obtained from HIV-negative patients and HIV-positive patients did not differ in the susceptibility to three antifungal compounds, including 5FC , FLU  and VOR . No significant differences were observed for ITR , POS , and ISA  when clinical and environmental isolates were compared. When all isolates were considered, the broadest ranges and highest MIC values were those of 5FC (<0.063 to >64 µg/ml), followed by FLU (0.125 to 32 µg/ml). The lowest MIC range was observed for ISA (<0.016 to 0.125 µg/ml), followed by POS, VOR and ITR (<0.016 to 0.5 µg/ml) (90 = 4 µg/ml) and ISA the highest (MIC90 = 0.063 µg/ml)  . Five is5 µg/ml) . Among t3 µg/ml) .C. neoformans var. grubii isolates Microsatellites are an excellent tool to discriminate among C. neoformans var. grubii isolates differed widely among the populations from the different countries. This finding supports the hypothesis that local geographic differences occur in Asia between C. neoformans var. grubii populations that could be the result from different founder effects and/or regional factors such as differences in the local environment and climate C. neoformans var. grubii compared cryptococcal isolates from Thailand with a globally collected set of isolates. This study showed that the Asian population was genetically less diverse than those occurring in Africa and North America C. neoformans var. grubii may disperse between China and Thailand The genotypic structure of the Isolates belonging to MC8 contained mainly cryptococcal isolates from HIV-infected patients, whereas MC2 and MC16 comprised mostly isolates from non-HIV-infected patients . A recenIn Japan and Thailand, environmental isolates co-occurred with clinical isolates in MC2, MC8, and MC16 , and theC. neoformans isolates to seven antifungals yielded no change in MIC ranges and MIC50 and MIC90 values of AMB and FLU when compared to previous studies 50 and MIC90 values for ISA were lower than those observed for the other antifungal agents, thus corroborating previous observations Susceptibility analysis of 493 C. neoformans, especially in MC17 and in Indonesia. Intrinsic resistance to 5FC in C. neoformans is uncommon and occurs with a low incidence of approximately 1–2% as reported from the USA in the 1990s C. neoformans may develop resistance to FLU after treatment Importantly, resistance in approximately 4% of the isolates to 5FC was observed. Most 5FC-resistant isolates came from Indonesia and Thailand where this drug is not in use Five out of 10 FLU resistant isolates  from Indonesia were also not susceptible to 5FC. Our findings suggested that isolates in certain MCs, especially MC17, may be prone to a decreased susceptibility to antifungals, especially FLU and 5FC. The observed double resistance to 5FC and FLU has not been reported before, and may pose a risk for the patients infected with such isolates. Isolates from HIV-infected and non-HIV-infected patients did not differ in MIC values of 5FC, FLU, and VOR, but the MIC ranges of isolates from HIV-infected patients were broader than those of isolates from non-HIV-infected patients. The highest MIC values of isolates from HIV-infected patients were 16 times higher for 5FC and FLU and two to four times as high as those from non-HIV-infected patients, respectively.in vitro antifungal susceptibility and genotyping of clinical and environmental isolates of C. neoformans from several Asian countries. Based on the in vitro susceptibility test results, the patients in the region seem to receive appropriate antifungal therapy with AMB plus 5FC for induction therapy followed by consolidation/maintenance therapy of FLU. AMB is still the most effective agent to treat an infection with C. neoformansC. neoformans, and therefore, initial treatment with AMB remains the recommended choice. However, given the well-tolerated nature and a excellent activity against Cryptococcus strains, the new generation of triazoles may become an important addition to the currently used antifungals.This is the first extensive report on C. neoformans populations from the Asian countries studied. Most of the countries had a unique distribution of MCs but the overall genetic diversity estimated by Simpson's index of diversity was high. Good in vitro antifungal activity was observed, but 5FC and FLU resistant, as well as 5FC/FLU double resistant isolates occurred.In summary, genotypic differences in microsatellite patterns occur between Table S1Origin of Cryptococcus neoformans var. grubii isolates and clinical background information of the patients.(DOC)Click here for additional data file.Table S2Environmental Cryptococcus neoformans var. grubii isolates from Thailand and Japan.(DOC)Click here for additional data file.Table S350, MIC90, and geometric mean for clinical isolates of The MIC range, MICC. neoformans in each country.(DOC)Click here for additional data file.Table S450, MIC90, and geometric mean for the different microsatellite complexes (MCs) of The MIC range, MICC. neoformans.(DOC)Click here for additional data file.


AbstractHemisorubim platyrhynchos is a medium- to large-sized pimelodid catfish distributed along several river basins of the Neotropical Region, noteworthy for representing an important fishery source. In this work, Hemisorubim platyrhynchos from three isolated populations were cytogenetically analyzed. The karyotype shows a diploid number of 2n=56 chromosomes comprising 22m, 16sm, 10st, 8a (FN=104). NORs detected by AgNO3 were located in the terminal regions of the short arm of a st chromosome pair, as confirmed by CMA3 and FISH using an 18S rDNA probe. C-banding revealed a small amount of heterochromatin in chromosomes, including the NORs, and one biarmed pair that showed conspicuous positive bands on both arms. This fact was also evidenced when using other banding techniques, such as RE (AluI), and indicates that this pair constitutes a species-specific cytogenetic marker. Hemisorubim platyrhynchos , popularly called “jurupoca” or porthole shovelnose catfish, is a pimelodid fish inhabiting the deeper and slow-moving sections of large South American rivers  has been cytogenetically studied and has had its diploid number, AgNORs location and C-banding reported  and 5 females (f), caught in the Parana River  and 2 specimens of undetermined sex from the Miranda River  were cytogenetically analyzed. The sampling sites in the Paraná River were: Ituzaingó (2 m), Itá Ibaté (2 f - 3 m), Yahapé (1 m), Puerto Abra (1 f), and Corrientes (2 f / 2 m) (Corrientes Province). Mitotic chromosome preparations were obtained according to the technique described by PageBreakthe Instituto de Ictiología del Nordeste, Universidad Nacional del Nordeste/Argentina. Nucleolus organizer regions (Ag-NORs) were revealed by the silver-staining method (Alu. I was used a concentration of 0.3 U/μL and the chromosome preparations were treated at 37°C for 4h. Chromosome staining with Chromomycin A3 (CMA3), a C-G specific fluorochrome, was applied according to the description provided by in situ hybridization (FISH) was carried out by means of biotinylated 18S rDNA probes (segment with 1700 pb) obtained from the nuclear DNA of the fish Oreochromis niloticus labeled with biotin-14-dATP by nick translation (Gibco cat Nº 18247-015), according to the manufacturer’s instructions. The hybridization technique, post-hybridization washes and visualization were carried out as reported by Fifteen specimens of g method  and C-baHemisorubim platyrhynchos presented the same results. The diploid number obtained was 2n=56 and the karyotype was composed of 22m+16sm+10st+8a (NF=104) without chromosomal differences between sexes  pair . The bri3 . This chong arms . The Alu pattern . The menHemisorubim platyrhynchos was composed of 22m+16sm+10st+8a (NF=104), however, despite having the same diploid chromosome number 2n=56, Hemisorubim platyrhynchos from the Paraná River/Brazil reported by Pimelodidae, 23 of the 27 PageBreakkaryotyped species have a diploid number of 2n= 56 chromosomes, except for Calophysus macropterus Lichtenstein, 1819, Luciopimelodus pati , and Pinirampus pirinampu  with 2n=50 and Megalonema platanum  with 2n=54, which seem to share other characteristics  and Pinirampus tigrinus  PageBreak (Steindachneridion sp. (=melanodermatum)  , Pinirames, 1840), Zungarot, 1821) , Steindao, 1918) and Steino, 2005) .3 staining and FISH with 18S rDNA exhibited fluorescent signals that correspond to the AgNOR sites , and the cytogenetic data confirm that the analyzed species belongs to the second group, because it has 2n=56 chromosomes, a high NF and the NORs localized on one single chromosome pair in the terminal position of the short arms, as it occurs with the other species of this group (Hemisorubim platyrhynchos shows a marked conservatism in its basic karyotype macrostructure, differing from many species of the Pimelodidae family, i.e., the “Pimelodus group”, which presents a wide karyotypic variability, even within the same hydrographic basin and within the same river.According to cytogenetic traits, this family could be divided into two: the “is group . Thus, d


Previous studies have shown that deep cerebellar nuclei (DCN)-lesioned mice develop conditioned responses (CR) on delay eyeblink conditioning when a salient tone conditioned stimulus (CS) is used, which suggests that the cerebellum potentially plays a role in more complicated cognitive functions. In the present study, we examined the role of DCN in tone frequency discrimination in the delay eyeblink-conditioning paradigm. In the first experiment, DCN-lesioned and sham-operated mice were subjected to standard simple eyeblink conditioning under low-frequency tone CS  or high-frequency tone CS  conditions. DCN-lesioned mice developed CR in both CS conditions as well as sham-operated mice. In the second experiment, DCN-lesioned and sham-operated mice were subjected to two-tone discrimination tasks, with LCS+ (or HCS+) paired with unconditioned stimulus (US), and HCS− (or LCS−) without US. CR% in sham-operated mice increased in LCS+ (or HCS+) trials, regardless of tone frequency of CS, but not in HCS− (or LCS−) trials. The results indicate that sham-operated mice can discriminate between LCS+ and HCS− (or HCS+ and LCS−). In contrast, DCN-lesioned mice showed high CR% in not only LCS+ (or HCS+) trials but also HCS− (or LCS−) trials. The results indicate that DCN lesions impair the discrimination between tone frequency in eyeblink conditioning. Our results suggest that the cerebellum plays a pivotal role in the discrimination of tone frequency. Eyeblink conditioning is one form of associative learning, which develops by the paired presentation of a neutral conditioned stimulus (CS), such as tones, and an unconditioned stimulus (US), such as electric shocks and air puffs. In delay conditioning the CS and US overlap temporally, although CS onset precedes US onset. The neuronal circuit for delay eyeblink conditioning has been extensively examined in rabbits, and the cerebellum is critical for conditioning in many species Recent developments and the availability of a variety of genetically-modified mice have made it possible to examine the role of certain neuronal cells and target molecules in the cerebellum that are specifically involved in eyeblink conditioning. However, genetically modified mice with altered cerebellar functions constantly result in the incomplete loss of conditioned responses (CR) during delay eyeblink conditioning Recently, the cerebellum was reported to be critical for not only motor coordination but also higher cognitive functions in mice, rats, and humans In the present study, we focused on the discrimination of tone frequency in the eyeblink-conditioning paradigm and examined the role of DCN in this particular paradigm. DCN-lesioned and sham-operated mice were subjected to two-tone discrimination tasks: 1) the low-frequency tone CS  and 2) the high-frequency tone CS . In the two-tone discrimination task, a mouse is demanded to make CR with one CS (the CS+) that is paired with US, but not with different CS (the CS–) that is presented without the US. This task has two aspects, the acquisition of CR to one CS and the inhibition of CR to the other CS. Therefore, two-tone discrimination is more complicated associative learning than simple eyeblink conditioning. Our results indicate that the cerebellum is essential for the acquisition of more complicated tasks in eyeblink-conditioning paradigms, such as two-tone discrimination tasks.Animal experiment protocols were approved and performed in accordance with the regulations outlined by the Japanese law and with the guidelines of Animal Use and Care Committee in Tsukuba University and Tokyo Metropolitan Institute of Gerontology.Male C57BL/6J mice aged 8–9 weeks old were used in all experiments. They were bought from a commercial breeder  1 week before experimentation. Mice were kept under standard housing conditions with a 12-h light/dark cycle (lights on at 7∶00 A.M.) as previously described Mice were housed individually 1 week before surgery. Animals were anesthetized with a cocktail of ketamine (80 mg/kg) and xylazine (20 mg/kg) and fixed to a stereotaxic apparatus. Each mouse received surgery for electrolytic lesions . Stereotaxic coordinates for lesions were based on the atlas of the mouse brain Mice were implanted with four electrodes  attached to the left upper eyelid: Two were used to record the electromyogram (EMG) from the orbicularis oculi and to deliver electric shocks to periorbital tissues. Four wires were soldered onto a four-pin connector, which was attached to the skull using dental cement. Eyeblink responses were recorded with an EMG instrument An eyeblink-conditioning system (EB-M4) was obtained from O’Hara and Co., Tokyo, Japan, and the conditioning was conducted as reported previously Two types of conditioned stimuli (CS) were used, LCS  and HCS , in combination with the unconditioned stimulus (US), an electric shock . The inter-stimulus interval of CS and US was 500 ms . The US After habituation sessions, the animals were subjected to delay eyeblink conditioning for 7 days. The LCS was used in Exp. 1a  , and HCSAfter habituation sessions, animals were subjected to two-tone discrimination tasks for 10 days. Daily acquisition training sessions of 50 trials were grouped into five blocks. Each block included five LCS and five HCS trials, and a total 25 LCS trials and 25 HCS trials were conducted in a daily session. In Exp. 2a , the LCS preceded and was co-terminated with the US, including one LCS only trial in each block, and the HCS always occurred alone without the US . Each CSEMG signals were band-pass filtered between 0.15 and 1.0 kHz, and fed into a computer at a sampling rate of 10 kHz. The frequency of CR occurrence was calculated from the eyelid EMG activity using the same analysis methods described by Sakamoto and Endo In Exp. 1, the threshold (mean plus SD of the EMG amplitudes) was calculated from the EMG activity amplitudes during the baseline period (300 ms before CS onset) in 50 trials. For each trial, we calculated the CR, spontaneous eyeblink responses (SER) and startle eyeblink responses (STR) based on baseline values. SER% and STR% were calculated for each session. CR% was calculated by subtracting the frequency of the SER from that of the CR in a daily 50-trial session (CR% = (CR- SER)/50×100), and was used as the learning index. We used this CR% to exclude unlearned responses  and to analyze the pure learning component, according to Sakamoto and Endo Normalized EMG amplitudes were calculated from EMG amplitudes of valid trials, which were obtained by measuring the EMG 300 ms before CS onset in each mouse, and then averaging these measurements for each group of mice in Exp. 1 In Exp. 2, experimental mice were subjected to the rotor rod test for 6 days before the two-tone discrimination task. The method for the rotor rod test was the same as previously described After behavioral experiments, mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS) containing 4% paraformaldehyde. The brains were removed from the skulls, and post-fixed overnight in PBS containing 4% paraformaldehyde. Coronal sections (50 µm) were cut and stained with cresyl violet. The lesioned area was examined under a microscope.post hoc analyses. For EMG analyses in eyeblink conditioning, the Mann-Whitney U test was used. P<0.05 was regarded as significant for all tests.Statistical analyses were performed using StatView 5.0 and Js-STAR. Two-way repeated measures ANOVA were used to analyze eyeblink conditioning and rotor-rod test data. The simple main effect test and Bonferroni test were used for In the present study, we used two types of tone-induced conditioned stimuli  and conducted delay eyeblink conditioning.F  = 3.20, P = 0.099) and the interaction of group×session  = 1.55, P = 0.17) was not significant, but that the main effect of the session was significant  = 33.73, P<0.01). These results indicate that the DCN lesions did not affect the acquisition of eyeblink conditioning with LCS.In eyeblink conditioning with the LCS , both DCF = 21.44, P<0.001), and this indicated that SER% in DCN-lesioned mice was lower than that in sham-operated mice. STR% in sham-operated and DCN-lesioned groups changed similarly, and no significant differences were observed in STR% between groups  = 0.90, P = 0.36).U test, U = 19.0, P = 0.48). The results show that DCN lesions did not have influence on EMG amplitudes during the CR period. Normalized EMG amplitudes in both groups increased between 50 and 100 ms from CS onset, and the short latency responses (SLRs) that are cerebellum-independent components were observed F  = 2.15, P = 0.17) and session  = 1.83, P = 0.11) as well as the interaction between group and session  = 0.89, P = 0.51) were not significant. The results indicate that the DCN lesions did not affect the acquisition of eyeblink conditioning with HCS.In eyeblink conditioning with HCS , both DCF = 13.27, P<0.01), and this indicates that SER% in DCN-lesioned mice was higher than that in sham-operated mice. STR% in sham-operated and DCN-lesioned groups changed similarly, and no significant differences were observed in the STR% between groups  = 1.45, P = 0.26).U test, U = 14.0, P = 0.52). The results indicate that DCN lesions did not have an influence on EMG amplitudes during the CR period. Normalized EMG amplitudes in both groups increased between 50 and 100 ms from CS onset, and SLRs were clearly observed.In Exp. 1, both DCN-lesioned and sham-operated mice learned standard delay eyeblink conditioning with LCS and HCS. Our previous results also suggest that the cerebellum is not so critical for mouse eyeblink conditioning when the salient CS conditions were used for conditioning F = 0.96, P = 0.36) were not significant, and that the main effects of session  = 3.01, P<0.01) and interaction of tone×session  = 2.03, P = 0.050) were significant. In the post hoc test, CR% in LCS+ trials significantly increased across the training sessions  = 4.88, P<0.01), CR% in session 9, 10, 11, 12 was larger than that in session 3 (P<0.05). Furthermore, CR% in LCS+ trials was larger than that in HCS− at session 9, 10, 11, 12 although they are not statistically significant. These results suggest that sham-operated mice discriminated the LCS+ and HCS− to acquire the CR.In Exp.2a, LCS (1 kHz) was paired with the US of an electrical shock, and the HCS (10 kHz) was presented alone . In shamF = 2.07, P = 0.20), sessions  = 1.90, P = 0.072) and interaction of tone×sessions  = 0.39, P = 0.93) were not significant. The results indicate that DCN-lesioned mice did not discriminate between LCS+ and HCS−. DCN-lesioned mice seemed to express eyeblink responses regardless of CS frequency (with or without the US).In contrast, in DCN-lesioned mice, CR% in LCS+ trials (16.4±8.6% –46.8±12.3%) were smaller than those in HCS− trials (42.5±8.7% –52.2±10.9%) throughout the sessions . Two-wayU test, U = 16.0, P = 0.093). In DCN-lesioned mice, no significant differences were observed in EMG amplitude between LCS+ and HCS− trials at the CR detection period . EMG amplitude also indicated that DCN-lesioned mice did not discriminate between LCS+ and HCS−.To examine the onset and peak latency of CR in LCS+ trials and HCS− trials, we analyzed normalized EMG amplitude for 500 ms from CS onset to US onset. F  = 167.85, P<0.01) and sessions  = 12.40, P<0.01) as well as the interaction of groups×sessions  = 4.74, P<0.01) were significant. In the post hoc test, fall latency in DCN-lesioned mice was smaller than that in sham-operated mice at all sessions (Ps <0.01).Rotor rod tests were conducted before eyeblink conditioning experiments to examine motor learning and coordination. F  = 15.34, P<0.01); however, the main effects of sessions  = 1.74, P = 0.10) and interaction of tone×sessions  = 0.54, P = 0.84) were not significant. These results indicate that sham-operated mice could discriminate between HCS+ and LCS− to acquire the CR.In Exp. 2b, HCS (10 kHz) was paired with electrical shock (US), and LCS (1 kHz) occurred alone . Sham-opF  = 2.27, P = 0.19) and the interaction of tone×sessions were not significant  = 1.77, P = 0.10), and the main effect of sessions was significant  = 3.16, P<0.01). These results indicate that DCN-lesioned mice did not discriminate between HCS+ and LCS−. Individual CR% in DCN lesion ned mice were examined, because the error bars in the LCS− trails were relatively large in Exp 2b. Two DCN lesioned mice showed high CR% (57–100%) at session 4 to10 in the LCS− trials  and LCS− trials (4.8±10.7% –47.8±14.1) throughout the sessions . In DCN-− trials .U test, U = 8.0, P<0.05); however, no significant differences were observed in EMG amplitudes between HCS+ and LCS− trials in DCN-lesioned mice . These results in EMG amplitude also indicate that sham-operated mice discriminated between HCS+ and LCS−, but not DCN-lesioned mice.To examine the onset and peak latency of CR in HCS+ trials and LCS− trials, we analyzed normalized EMG amplitude for 500 ms from CS onset to US onset. F  = 169.30, P<0.01) and sessions  = 12.77, P<0.01), and the interaction of groups×sessions  = 5.09, P<0.01) were significant. In the post hoc test, fall latency in DCN-lesioned mice was smaller than that in sham-operated mice at all sessions (Ps <0.01).In the rotor rod tests , sham-opDCN lesions were verified after all behavioral tests according to Sakamoto and Endo In our previous studies, both DCN-lesioned and sham-operated mice acquired the CR in delay eyeblink conditioning under salient CS conditions  In Exp. 1, DCN-lesioned and sham-operated mice were subjected to simple eyeblink conditioning under a LCS or HCS. Both sham-operated and DCN-lesioned mice acquired the CR in eyeblink conditioning under LCS  conditions  and HCS DCN-lesioned mice were subjected to two-tone discrimination tasks in Exp. 2. In Exp. 2a, 1 kHz CS (LCS+) was paired with the US but 10 kHz was not (HCS−) during eyeblink conditioning . CR% in According to behavioral audiograms in the C57BL/6J mouse, optimal tone frequencies for the normal mouse are 8–32 kHz We found for the first time that the cerebellum is critical for the discrimination of tone frequency in eyeblink-conditioning paradigms. We revealed that in mice the amygdala plays an important role in eyeblink conditioning but not the cerebellum under salient CS The two-tone discrimination task and the reversal task have been conducted to examine the cognitive function in the hippocampus We demonstrated that DCN plays an important role in the two-tone discrimination task , then, wpcd) mutant mice nervous mutant mice Recently, the cerebellum has been reported to be critical not only for motor coordination and learning but also for cognitive function in animals, including humans In summary, we revealed that DCN play an important role in the discrimination of tone frequency in eyeblink conditioning paradigms. This finding suggests that the cerebellum is involved in complicated associative learning. The role of the cerebellum on other psychological functions, for example, socio-emotional function such as emotionality, communication, and social interaction, need to also be examined in the future.


DGAT1) is a functional and positional candidate gene for milk and intramuscular fat content. A bovine DGAT1 overexpression vector was constructed containing mouse MCK promoter and bovine DGAT1 cDNA. MCK-DGAT1 transgene in FVB mice was researched in present study. The transgene DGAT1 had a high level of expression in contrast to the endogenous DGAT1 in posterior tibial muscle of the transgenic mice, but a low expression level in the cardiac muscle. Compared with wild-type mice, triglyceride and DGAT1 content were approximately fourfold and 50% increased in posterior tibial muscle of the transgenic mice, respectively, while a little increase in cardiac muscle.The gene encoding diacylglycerol acyltransferase ( DGAT1) encodes acyl CoA:diacylglycerol acyltransferase (DGAT) which catalyzes the terminal and only committed step in triacylglycerol synthesis [DGAT1 spaning 14117 base pairs (bp) was assigned to a region on bovine chromosome 14 close to the centromere and contains 17 exons and 16 introns [et al. [DGAT1 as a positional and functional candidate gene for intramuscular fat deposition in cattle. Myocellular overexpression of DGAT1 resulted in increased intramyocellular TG levels but decreased myocellular DAG and ceramide levels [Triacylglycerols (TG) are quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase 1  is critical to the energy metabolism of skeletal muscle as the key enzyme in the phosphorylcreatine shuttle between mitochondria and the myofibrils . The MCKet al.  demonstret al. . Besideset al. . Long te [et al.  to increDGAT1 could express in skeletal muscle of mice under the control of mouse MCK promoter, and determine the effect of bovine DGAT1 overexpression on triglyceride synthesis and storage in MCK-DGAT1 transgenic mice.In the present study, we wanted to investigate whether bovine We used the 1.38 kb mouse MCK promoter, which is widely used for skeletal and cardiac muscle transgene expression, to direct the expression of a full-length bDgat1 cDNA. The RT gene expression analysis was performed on five transgenic mice and five WT mice, which showed that expression of the transgene was primarily restricted to skeletal muscle, and had a high level contrast to the endogenous DGAT1 in posterior tibial muscle of the transgenic mice  and WT (20.03 ± 1.31 g) mice. TG levels in posterior tibial muscle were approximately fourfold increased in MCK-DGAT1 mice compared with WT mice , also subjectively assessed as marbling, represents an important beef quality trait . The MCKet al. [Liu et al.  first deThe transgene contains, from the 5′-end to the 3′-end, an 1.38 kb mouse MCK promoter, and an 1.48 kb full-length bovine DGAT1 (bDGAT1) cDNA  mice were purchased. Mice were housed in a barrier facility with 12-hour light/12-hour dark cycles and fed normal chow.Total RNA was extracted from cardiac and posterior tibial muscle using TRIzol Reagent purchased from Invitrogen. The muscle were first homogenized with TRIzol Reagent, then RNA was separated in chloroform, precipitated by isopropanol, and washed by 75% ethanol, lastly the RNA was dissolved in RNase-free water. Total RNA was reverse-transcribed into cDNA which was used in the subsequent PCR amplification. The mRNA levels were quantified using the following primers: mouse endogenous DGAT1, forward, 5′- GCCCAAGGTAGAAGAGGACGAG-3′, reverse, 5′- AGTATGATGCCAGAGCAAACACG-3′, PCR temperature program, 95°C 3 min, then 95°C 30 s, 59°C 30 s, 72°C 30 s for 30 cycles, 72°C 5 min; transgene DGAT1, forward, 5′- AGAGGAGGTGCGGGATGTGG-3′, reverse, 5′- TCGCGGTAGGTCAGGTTGTCG-3′, PCR temperature program, 95°C 3 min, then 95°C 30 s, 61°C 30 s, 72°C 30 s for 30 cycles, 72°C 5 min. mRNA levels were then expressed as ratios relative to Gapdh, which express stably in this test, forward, 5′- GATGCCCCCATGTTTGTGAT-3′, reverse, 5′- GGGTGGTCCAGGGTTTCTTA-3′, PCR temperature program, 95°C 3 min, then 95°C 30 s, 54°C 30 s, 72°C 30 s for 30 cycles, 72°C 5 min. The RT gene expression analysis was performed on five transgenic mice and five WT mice.Tissues were homogenized by pestles in isoamyl alcohol. Triglyceride mass in lipid extracts was enzymatically determined with a colorimetric kit.Tissues were homogenized by pestles in PBS. Total DGAT1 content was measured with a DGAT1 ELISA kit. Double antibody sandwich ELISA is used in this kit to measure the content of DGAT1 in tissue homogenate quantitatively.Data were expressed as means ± SD. Statistical differences were analyzed by ANOVA to determine overall treatment effects including TG and DGAT1 content (SPSS 11.0). A two-tailed P value of 0.01 was used to indicate statistical significance.The experiment was conducted according to protocols approved by the Institutional Animal Care and Use Committee at Tianjin Agriculture University.The authors declare that they have no competing interests.FY carried out the whole experiment of the studies and drafted the manuscript. ZW, GL, HG and GS participated in the design of the study. XD, XL and XG participated in the experiment and helped to the data analysis. All authors read and approved the final manuscript.Zhuying Wei Joint first author.


Multicentric Castleman disease (MCD) is a lymphoproliferative disorder caused by human herpesvirus 8 (HHV8) infection HIV associated MCD (HIV-MCD) presents with various clinical symptoms. Many HIV-negative MCD patients are often treated with anti-human interleukin-6 (IL6) receptor monoclonal antibodies (tocilizumab), and successful results have been reported. IL-6 plays an important role in the development of both HIV-positive and HIV-negative MCD; however, the efficacy of tocilizumab in HIV-MCD patients is unknown. We herein report the clinical and biologic courses of two HIV-MCD patients treated with tocilizumab. In both cases, a significant and rapid clinical improvement was observed after the first infusion. However, the treatment efficacy was not maintained for a long period, and relapse occurred at 15 and 22 weeks, respectively. Both patients received rituximab and subsequently achieved complete clinical remission. Our report, in addition to data presented in the literature, suggests that tocilizumab could be an initial treatment option in patients with HIV-MCD. Multicentric Castleman disease (MCD) is a lymphoproliferative disorde, and HIV-associated MCD (HIV-MCD) is caused by human herpesvirus 8 (HHV8) infection in HIV-positive patients . HIV-MCD4/μ, albumin 3.8 g/dl and CRP 0.15 mg/dl.A 44-year-old male who was HIV-1 seropositive for several years and did not start treatment with combination antiretroviral therapy (cART), with a CD4 cell count of 188 cells/μl and a viral load of 74 copies/μl, was diagnosed with Kaposi’s sarcoma and treated with two cycles of liposomal doxorubicin and cART. Hepatitis C and B were negative. Eight months after being diagnosed with Kaposi’s sarcoma, he presented with a high fever, fatigue and lymph nodes swelling throughout his body. Blood tests revealed anemia (hemoglobin: 8.3 g/dl), thrombocytopenia (3.3×104/μ), a low albumin level (2.3 g/dl) and a high CRP level (10.75 mg/dl). The high fever persisted for two weeks. A lymph node biopsy demonstrated remarkable infiltration of polyclonal plasma cells and plasmablastic cells in the interfollicular areas. Lymph node architecture was retained. Vascular proliferation was observed between the follicles, with perivascular hyalinization. The levels of HHV8 and human IL6  in the blood were 460,000 copies/μl and 41.7 pg/ml, respectively. The patient was diagnosed with HIV-MCD and 8 mg/kg of tocilizumab was administered intravenously. The persistent high fever disappeared within a few hours. There were no adverse events of tocilizumab treatment. After one week, the laboratory abnormalities recovered: hemoglobin 10.8 g/dl, platelets 11.2×104/μ, an albumin level of 2.1 g/dl, a CRP level of 8.18 mg/dl, an HHV8 titer of 3,400,000 copies/μl and a hIL6 level of 305 pg/ml, indicating HIV-MCD relapse. A second lymph node biopsy showed angiofollicular hyperplasia and interfollicular plasma cell infiltration. HHV8 antigens were more strongly positive in lymphocytes than that observed on the first biopsy. The patient received tocilizumab infusions once in week for two weeks (the 15th and 16th weeks); however, his symptoms and blood test abnormalities worsened. Tocilizumab was discontinued and he recovered following the administration of four cycles of rituximab treatment. He has since remained in remission for four years.The HHV8 concentration and hIL6 level in the blood decreased to 120 copies/μl and 18.2 pg/ml, respectively, after treatment  and hyper gammaglobulinemia . He was diagnosed with HIV-MCD based on the findings of a lymph node biopsy. The follicles showed varied degrees of involution and hyalinization of the germinal centers with prominent mantle zones that intruded into the germinal centers, completely effacing them. In the mantle zone cells, variable numbers of plasma cells and lymphocytes with HHV8 antigens were observed. The levels of hIL6 and HHV8 in the blood were 53.5 pg/ml and 390,000 copies/μl, respectively. The patient received 8 mg/kg of tocilizumab 10 days after appearance of symptoms. Following the administration of tocilizumab, the high fever immediately decreased. The abnormal values on the blood examinations improved two weeks after treatment: hemoglobin 14.8 g/dl, platelets 12.9×104/μl, albumin 4.4 g/dl and CRP 0.07 mg/dl. The levels of HHV8 and hIL6 decreased to 5,100 copies/μg and 35 pg/ml, respectively, eight weeks after treatment , although hepatitis B was negative. In 2012, he presented with a high fever and fatigue with swollen lymph nodes throughout his body. A blood test showed anemia (a hemoglobin level of 6.1 g/dl), a low albumin level (2.4 g/dl), a high CRP level (13.59 mg/dl), a low platelets count  and will assist in elucidating these issues. The accumulation and analysis of case reports is required to evaluate when and how tocilizumab should be administered with or without other drugs, including rituximab, ganciclovir and etoposide, in patients with HIV-MCD.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.IL6: Anti-human interleukin-6; HIV-MCD: HIV associated multicentric Castleman disease; HHV8: Human herpesvirus 8; CRP: C-reactive protein; cART: Combination antiretroviral therapy; hIL6: Human IL6; vIL6: Viral IL6.Although Hanabusa H. has received honoraria from Baxter Healthcare, NovoNordisk, Bayer, Pfizer, and KaketsuKen; and has served on advisory boards for Baxter Healthcare (limited to hemophilia care), he has no competing interest related with this case report (Tocilizumab and Chugai Co.). Nagao and Nakazawa declare that they have no competing interests.AN, SN and HH cared for the patients. AN wrote the manuscript. HH designed the study and helped to draft the manuscript. All authors have read and approved the final manuscript.


A novel mouse model that eliminates cholinergic neurotransmission in the striatum while leaving glutamate release intact reveals differential effects on cocaine-induced behavior and dopaminergic responses. Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease. The neurotransmitters dopamine and acetylcholine play opposite roles in the striatum (a brain region involved in motor control and reward-related behaviour), and their balance is thought to be critical for striatal function. Acetylcholine in the striatum has been linked to a number of functions, including control of locomotor activity and response to drugs of abuse. However, striatal cholinergic interneurons can also release glutamate (in addition to acetylcholine) and it is presently unclear how these two neurotransmitters regulate striatal-dependent behaviour. Previous work has attempted to resolve this issue by ablating cholinergic neurons in the striatum, but this causes loss of both cholinergic and glutamatergic neurotransmission. In this study, we created a novel genetic mouse model which allowed us to selectively interfere with secretion of acetylcholine in the striatum, while leaving total striatal glutamate release intact. In these mice, we observed minimally altered behavioural responses to cocaine, suggesting that striatal glutamate, rather than acetylcholine, is critical for cocaine-induced behavioural manifestations. Furthermore, elimination of striatal acetylcholine release affects how striatal output neurons respond to dopamine, by up-regulating dopaminergic receptors and changing behavioural responses to dopaminergic agonists. Our experiments highlight a previously unappreciated physiological role of cholinergic-glutamatergic co-transmission and demonstrate how a population of neurons can use two distinct neurotransmitters to differentially regulate behaviour. The striatum is the major input gateway to the basal ganglia. Striatal activity plays important roles in controlling motor functions and goal-directed and reward-related behaviours In contrast to the widely known effects of dopamine in the striatum, we know considerably less about how ACh shapes striatal function. Cholinergic neurons form a small population of aspiny and large striatal interneurons that provide the sole source of cholinergic innervation to MSNs Elimination of cholinergic neurons in the striatum, using ablation strategies, indicated that these neurons have a role in regulating spontaneous and cocaine-induced locomotor activity, as well as its rewarding properties To investigate the possibility that cholinergic neurons can use these two distinct neurotransmitters differentially to regulate striatal circuitry, we generated a novel mouse line in which we selectively eliminated ACh release by deleting the VAChT gene in the striatum. Our results reveal specific roles for ACh release in regulating dopamine receptor-mediated locomotor responses, but suggest that some of the previous functions attributed to these neurons are related to their ability to release glutamate.flox/flox, flox/flox mice had normal levels of VAChT and other pre-synaptic cholinergic markers. In addition locomotor activity, grip-strength, and fatigue were identical in VAChTflox/flox mice and wild-type mice To address specific roles of ACh release in striatal function we generated a VAChT floxed mouse line (VAChTRosa26 reporter mice (Rosa26-YFP mice), in which the Rosa26 locus expresses YFP once Cre-mediated recombination has occurred (flox/flox mice to generate mice with selective elimination of VAChT in the striatum (VAChTD2-Cre-flox/flox) or control mice (VAChTflox/flox). Genotyping for these lines is shown in D2-Cre-flox/flox mice were born in the expected Mendellian ratio and did not present overt phenotypes. We found no gross morphological alterations in the striatum or other brain sections stained with hematoxylin/eosin in VAChTD2-Cre-flox/flox mice compared to control mice (unpublished data).In order to selectively eliminate VAChT in the striatum, we used the D2-Cre bacterial artificial chromosome (BAC) transgenic mouse line generated by GENSAT occurred . We founoccurred . We did occurred . Partialoccurred . We therD2-Cre-flox/flox. As expected, based on the observations with the D2-Cre;Rosa26-YFP mice, both mRNA and protein levels for VAChT were almost abolished in the striatum of VAChTD2-Cre-flox/flox  = 1.702, p = 0.095) or fatigue  = 49, p = 0.710). Interestingly, we also found that relative to controls, VAChTD2-Cre-flox/flox mice showed no deficit in motor performance or motor learning assessed using the rotarod test  = 0.0000409, p = 0.995; both sets of mice improved their performance, F = 41.614, p<0.001; and there was no interaction between genotype and session, F = 1.333, p = 0.220). These results show that despite a decrease in the levels of VAChT in the spinal cord there were no detectable changes in motor function. The rotarod experiments also suggest that VAChTD2-Cre-flox/flox mice are physically fit and that motor learning does not appear to depend on striatal cholinergic activity.Because we detected the presence of Cre-mediated recombination in motoneurons in the spinal cord , which clox mice . HoweverD2-Cre-flox/flox mice performed identically to controls, suggesting that important cognitive functions are preserved in this new mouse line  = 0.651, p = 0.431, a significant effect for object, F = 21.559, p<0.001 and no Object × Genotype interaction, F = 0.0185, p = 0.893).Next, as a further control experiment, we determined if the elimination of ACh release in the striatum could interfere with cognitive performance that is believed to be generally independent of striatal function. We used object recognition memory, a task that is thought to be dependent on the hippocampus Previous experiments have shown that the density of cholinergic neurons in the accumbens, as well as expression of ChAT, is decreased in the post-mortem brain of schizophrenic individuals D2-Cre-flox/flox mice to controls (t(48) = 0.1464; p = 0.884) or counts of vertical activity  = 1.027; p = 0.315) were essentially identical in the two strains. Importantly, in control experiments D2-Cre mice did not differ in locomotor activity from respective wild type mice  = 0.321, p = 0.576, significant effect of time, F = 14.411, p<0.001 and no interaction Genotype × Time, F = 0.947, p = 0.591; total activity was not different, Mann-Whitney, T(29) = 213, p = 0.431). Finally, we also tested inter-session habituation by investigating locomotor activity in 3 consecutive days in the open-field  = 0.932, p = 0.342). The activity decreased over the day  = 10.244, p<0.001) and both genotypes habituated to the environment at comparable rates  = 1.506, p = 0.230). Evidently, deletion of VAChT in the striatum does not affect general spontaneous activity or compromise the capacity to habituate to a new environment.It has been shown that VGLUT3-null mice present hyperactivity, which was attributed to decreased ACh release from striatal cholinergic neurons due to decreased filling of synaptic vesicles with ACh en-field . We obseflox/flox mice and VAChTD2-Cre-flox/flox mice  = 6.531, p = 0.014, significant effect of treatment, F = 15.611, p<0.001, and no Genotype × Treatment interaction, F = 0.983, p = 0.381). There was no difference between the two genotypes in their ability to increase activity in response to cocaine-injected i.p. at 5 mg/kg dose  = 0.201, p = 0.661, significant effect of time, F = 12.820, p<0.001, and no Time × Genotype interaction, F = 1.373, p = 0.121). Paradoxically, at 20 mg/kg VAChTD2-Cre-flox/flox mice showed a smaller effect of cocaine in locomotor activity than controls  =  11.345, p<0.001, significant effect of time, F = 9.464, p<0.001, and no Time × Genotype interaction, F = 0.945, p = 0.537). Analysis of total activity counts showed a clear effect of genotype ((23) = 166.000, p<0.05). At 40 mg/kg both genotypes showed similar responses (t(14) = 0.980, p = 0.344), suggesting that lack of striatal VAChT altered the response to 20 mg/kg of cocaine, but overall did not cause increased sensitivity to locomotor effects of cocaine.Previous experiments in mice in which cholinergic interneurons were ablated suggested that decreased ACh levels increase sensitivity of mice to the locomotor effects of cocaine D2-Cre-flox/flox mice. We were unable to obtain reliable CPP with either genotype at 5 mg/kg of cocaine (unpublished data). In contrast, at 20 mg/kg we detected robust CPP in both genotypes  = 0.443, p = 0.521, significant effect of treatment, F = 86.033, p<0.001, and no Genotype × Treatment interaction, F = 0.0118, p = 0.916). In these experiments we used an extended protocol D2-Cre-flox/flox mice (unpublished data). In addition, neither extinction of CPP nor relapse, measured as a reinstatement of CPP by a priming injection of cocaine after extinction, were altered in mice without striatal VAChT  = 0.00057, p = 0.982, significant effect of treatment, F = 7.457, p = 0.029, and no Genotype × Treatment interaction, F = 9.67×10−5, p = 0.992). Therefore, there was no difference in CPP response for the two genotypes.Cocaine increases firing of striatal cholinergic neurons D2-Cre-flox/flox mice, but the relative increase in behavioural sensitization was not different between genotypes  = 4.902, p = 0.042, significant effect of treatment, F = 33.855, p<0.001, and no Genotype × Treatment interaction, F = 0.496, p = 0.491). Thus, elimination of striatal ACh release caused a small change in the dose-response profile of cocaine-treated mice in intermediate doses: a slight increase in activity is observed at 10 mg/kg, whereas a decrease in locomotor response is observed at 20 mg/kg in mutant mice.Behavioural sensitization protocols for cocaine likely reflect altered synaptic plasticity in response to the drug D2-Cre-flox/flox mice. For that, we first determined the concentration of dopamine and metabolites in the striatum of VAChTD2-Cre-flox/flox mice and compared these to control mice. In general there were no major changes in dopamine and metabolites in these mutant mice (p<0.001), suggesting potential relatively minor alterations in dopamine dynamics or metabolism.The balance between acetylcholine-dopamine is important in a number of conditions, including PD; therefore we further investigated dopaminergic function in VAChTant mice . HoweverD2-Cre-flox/flox mice compared to control mice (t(12) = 2.756, p<0.05, D2R, t(14) = 2.300, p<0.05). In contrast, D2R mRNA expression in the midbrain was not altered (t(12) = 3.678, p<0.05). Homozygous mice expressing D2-BAC-GFP construct present some dopaminergic phenotypes To further assess dopaminergic function, we performed qPCR analysis for D1R and D2R expression in the striatum. We detected an increase in the expression of D1R and D2R mRNAs in the striatum of VAChT altered . G-proteD2-Cre-flox/flox mice we initially performed Western blots. Unfortunately, we were unable to obtain a reliable D1 antibody that showed specific detection of D1R (unpublished data). However, we obtained a D2 antibody that labelled only one major band with the correct molecular mass (t(14) = −3.628, p<0.01). In order to provide an independent measure of D1R activity and test if D1-mediated responses would be altered in VAChT-eliminated mice, we used pharmacological magnetic resonance imaging (phMRI) phMRI is a variant of functional magnetic resonance imaging that indirectly detects neuronal activity using blood oxygenation level-dependent (BOLD) MRI signal changes D2-Cre-flox/flox mice following injection of SFK 81297 .To confirm the increased alteration of dopamine receptors in the striatum of VAChTlar mass . Quantif3 mg/kg, . The cha agonist , p<0.01.D2-Cre-flox/flox mice had significantly higher locomotor responses to two doses of SKF 81297  = 11.654, p<0.01, significant effect of drug concentration, F = 34.476, p<0.001, and significant Drug Concentration × Genotype interaction, F =  4.277, p<0.01, Tukey post hoc test showed significant differences with SKF 81297 doses of 3 mg/kg (p<0.01) and 8 mg/kg (p<0.001)). Moreover, VAChTD2-Cre-flox/flox mice also showed enhanced inhibition of locomotion in response to low doses of the D2R-selective agonist quinpirole  = 12.543, p<0.001, significant effect of drug, F = 42.223, p<0.001, but the interaction was not significant, F = 2.052, p = 0.092). Analysis of locomotion in response to the individual doses showed a significant difference for 0.005 and for 0.01 mg/kg quinpirole dose (p<0.01). Taken together, these data reveal important alterations in the expression and function of striatal dopamine receptors in VAChTD2-Cre-flox/flox mice.To test if the increased expression/sensitivity of D1R and D2R has direct behavioural consequences, we investigated the effects of the selective dopaminergic agonists SKF 81297 (D1R agonist) and quinpirole (D2R agonist) on locomotor activity. VAChTHere we present a series of evidence that delineates the role of released ACh from those of VGLUT3-dependent glutamate release from striatal cholinergic interneurons. Our data provide a novel perspective on the function of striatal cholinergic neurons suggesting the possibility that they can use distinct neurotransmitters to regulate striatal circuitry. We found that elimination of VAChT in the striatum, without disruption of VGLUT3, did not cause overt disruptions or alterations in several behavioural tasks previously thought to be dependent on striatal ACh release, such as motor learning, sensorimotor gating, and spontaneous locomotor activity. However, we uncovered a novel form of regulation of MSNs by cholinergic tone, and found that selective silencing of striatal ACh release results in an increase in the responses to D1R and D2R agonists. In contrast to the effects of direct dopamine receptor agonists, we found that overall these mice do not show increased locomotor response to cocaine. Similarly, sensitization and rewarding effects of cocaine did not seem to be dependent on striatal release of ACh. Thus, our results significantly depart from previous studies in which the specific contributions of striatal ACh release (mediated by VAChT) were not separated from those of glutamate release (mediated by VGLUT3). These data suggest that VGLUT-3 dependent glutamate release may influence locomotor activity and responses to cocaine considerably more than VAChT-dependent ACh release. Our data suggest that targeted approaches aimed at inhibiting VAChT activity in the striatum may potentially provide a novel strategy to enhance dopaminergic signalling, without causing other major behavioural disturbances.D2-cre-flox/flox mice indicated that elimination of ACh release in the striatum does not seem to play a major role in motor function and motor learning, at least for acrobatic motor skills in the rotarod test. This observation is also in agreement with previous experiments in striatal cholinergic neuron-ablated mice that presented no deficiency in rotarod performance D2-Cre-flox/flox mice and more sophisticated motor behavioural tests may be necessary to pinpoint possible roles for striatal ACh in motor learning and performance.Our studies in VAChTThere are multiple lines of evidence that pharmacological modulation of cholinergic receptors regulates locomotor activity. It is known that muscarinic antagonists increase locomotor activity and M1 and M4 muscarinic receptor KO mice are hyperactive Previous attempts to assess the function of cholinergic neurons in the striatum were performed following the ablation of cholinergic neurons using immunotoxin-mediated cell targeting. Injection of toxin targeting transgenic cholinergic neuron in the accumbens led to an 80% decrease in ChAT-positive neurons D2-Cre-flox/flox mice. Interestingly, recent data have shown that cholinergic neurons in the habenula secrete both ACh and glutamate (mediated by VGLUT1), and release of either of these neurotransmitters appears to depend on the frequency of stimulation While an optogenetic approach provides a novel paradigm to acutely activate or inactivate populations of neurons, it is unlikely that this method can separate VAChT from VGLUT3-dependent neurotransmission as selectively as that which can be achieved using VAChTD2-Cre-flox/flox mice. It is unlikely that 40% reduction in ACh release observed in VGLUT3-null mice can be responsible for the hyperactive phenotype. Indeed, independent mouse lines with a 50% decrease in VAChT expression, and concomitant reduction of ACh release D2-Cre-flox/flox mice, VGLUT3 floxed mice, and double knockouts will be necessary to provide an assessment of independent effects of VGLUT3-mediated neurotransmission in the striatum.The role of VGLUT3 in striatal function is far from being fully understood D2-cre-flox/flox mice may contribute to these phenotypes should also be taken into account. We did not detect Cre-expression in cholinergic neurons in the penduculopontine area, for example  were obtained from the GENSAT project via the mutant mouse regional resource centers. VAChTttc2, and a recent report suggests that homozygous D2-GFP mice, generated using the same BAC construct, are hyperactive and show a number of dopamine-related phenotypes ttc2. We confirmed that the D2-Cre indeed have increased expression of the TTC2 mRNA (unpublished data). However, heterozygous D2-Cre mice showed no locomotor phenotype. Moreover, these mice showed normal levels of D1R, D2R, and M4-muscarinic receptors Cos/J, stock number 006148) were obtained from Jackson Laboratories. Animals were housed in groups of three to four mice per cage without environment enrichment in a temperature-controlled room with 12-h light–12-h dark cycles, and food and water were provided ad libitum. Mouse stocks were SPF, however experimental subjects were kept in a conventional mouse facility.Rosa26-YFP mice (B6.129X1-Gt(ROSA)26SorAll studies were conducted in accordance with the NIH and the Canadian Council of Animal Care (CCAC) guidelines for the care and use of animals with approved animal protocol from the Institutional Animal Care and Use Committees at the University of Western Ontario (protocol number 2008–089). Only male mice were used for the behavioural studies, and they were at least 12 weeks old. Mice were randomly assigned to distinct experimental groups. Only mice used for evaluation of spontaneous locomotor behaviour were used in other tasks.For the immunofluorescence experiments we followed a protocol previously described 3H]methyl-choline, and the release of labelled ACh was determined essentially as described Slices were obtained from the striatum and hippocampus of control and test mice, labelled with [Striatal synaptosomes were prepared by the method of All behavioural experiments were performed between 9 a.m. and 4 p.m. in the light cycle, essentially as previously described The dissected brain tissues were homogenized in 0.2 M perchloric acid with 100 µM EDTA-2Na. Samples were spun in a microcentrifuge at 12,000 rpm for 15 min at 4°C. Samples of the supernatant were then analyzed for norepinephrine (NE), dopamine (DA), and its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) by NoAb BioDiscoveries .The HPLC used was an Eicom EP-700 with electrochemical detection (Eicom ECD-700). To elute catecholamines from the reverse phase column , a mobile phase consisting of 0.1 M citric acetate buffer pH 3.5 with 5 mg/ml EDTA-2Na, 220 mg/L sodium octane sulfonate, and 22% methanol was used.These experiments have been described in A Grip Strength Meter from Columbus Instruments  was used to measure forelimb grip strength essentially as described The rotarod task followed a previously described protocol The CPP protocol was modified from Behavioural sensitization was performed as described The general procedure was previously described, but for analysis we used Anymaze D2-cre/flox/flox, N = 5; control, N = 4) were anesthetized with 4% isofluorane and maintained at 1.5% isoflurane during the MRI scanning. Two intraperitoneal (I.P.) catheters  were used for injection of saline and SFK. The catheters were secured in place with subcutaneous sutures. Catheters were connected to polyethelyne tubing  and to syringes containing saline and SFK for remote injection during imaging. Mice were placed in a custom built frame designed to secure the skull and minimize respiration induced movement during image acquisition. Mice were imaged on a 9.4 Tesla small animal MRI scanner  equipped with a two-channel surface coil (diameter  = 2 cm). A fast low angle shot (FLASH) pulse sequence was used to acquire anatomical images . Respiratory gated lower resolution FLASH images were also acquired for pharmacological imaging  to measure blood oxygen level–dependent (BOLD) signal changes. Seven contiguous axial slices (500 μm thick) covered the brain. Each animal received two injections: first, an injection of 0.5 ml physiological saline (0.9%) administered over a 30 s period (control), and second, SFK 81297 (3 mg/kg), diluted in 0.5 ml physiological saline, also administered over a 30 s period (drug). For the control experiment, images were acquired for 8 min prior to saline injection and then for 20–50 min following injection. For the drug experiment, images were acquired for 8 min prior to drug injection and then for 80–180 min after injection. Throughout the imaging session, body temperature and respiration rate was monitored every 10 min using the MR-Compatible, Model 1025 monitoring system . Temperature was maintained at 37.5°C using a warm air blower, and respiration rate ranged from 45–66 (mean 54 BPM). Following imaging, mice were euthanized by cervical dislocation while still under isoflurane anaesthesia.Mice . For locomotion experiments we used ANOVA with repeated measures, and when appropriate, a Tukey post hoc comparison test was used. For pharmacological MRI, the area under the curve of the signal time course was compared between VAChTD2-cre/flox/flox mice and control mice using a Student's t test.Data are expressed as mean ± SEM. Sigmastat 3.1 software was used for statistical analysis. Comparison between two experimental groups was done by Student's Figure S1D2-Cre drives the expression of Cre in striatal cholinergic neurons. (a) Sections from different regions of the central nervous system were immunostained for CHT1 (Red) and YFP (Green) in D2-Cre;Rosa26-YFP mice. Arrows show localization of Cre expression (YFP) in cholinergic neurons (CHT1 staining). Arrowheads show cholinergic neurons that do not express Cre.(TIF)Click here for additional data file.Figure S2D2-Cre-flox/flox allele and identification by genotyping. (a) Cartoon representing the VAChT alleles before and after recombination. (b) PCR genotyping for VAChTflox/flox and VAChTD2-Cre-flox/flox mice. Lanes 1 and 2 WT PCR product, lane 3 flox/flox PCR product, and lane 4 heterozygous mice PCR product. (c) Lane 1 VAChTflox/flox, lane 2 VAChTD2-Cre-flox/flox, lane 3 VAChTD2-Cre-flox/flox, and lane 4 VAChTflox/flox genotyping.Representation of VAChT(TIF)Click here for additional data file.Figure S3D2-Cre-flox/flox mice.  Quantification of mRNA expression for VAChT, ChAT, and CHT1, respectively, in the Spinal cord.  Quantification of protein levels in the Spinal cord for VAChT, ChAT, and CHT1, respectively. Synaptophysin immunoreactivity was used to correct for protein loading between experiments. (g) Representative Western blot of VAChT, synaptophysin, ChAT, CHT1, and actin ** p<0.01 and *** p<0.001.Cholinergic parameters in the spinal cord of VAChT(TIF)Click here for additional data file.Figure S4D2-Cre-flox/flox mice. (a) Grip-force analysis of VAChTflox/flox and VAChTD2-Cre-flox/flox mice. (b) Time spent hanging upside-down from a grid, to measure fatigue for VAChTflox/flox mice and VAChTD2-Cre-flox/flox mice (cut-off time 60 s). (c) Motor learning and acrobatic motor skills of VAChTD2-Cre-flox/flox mice determined using the rotarod . (d) Object recognition memory in VAChTflox/flox and VAChTD2-Cre-flox/flox mice. * p<0.05.Motor function is not altered in VAChT(TIF)Click here for additional data file.Figure S5D2-Cre-flox/flox mice. (a) Short-term habituation of acoustic startle responses to 30 acoustic startle stimuli of 115 db white noise delivered every 20 s. (b) Prepulse inhibition of acoustic startle responses with different prepulse intensities and prepulse-pulse intervals as indicated.Sensorimotor gating is not altered in VAChT(TIF)Click here for additional data file.Figure S6Biochemical and behavioural parameters of heterozygous D2-Cre mice. (a) Horizontal activity as described in (TIF)Click here for additional data file.Table S1Percentage of co-localization between cell with Cre-induced recombination and CHT1.(TIF)Click here for additional data file.


Case-control studies of diabetes and adiposity included 15,326 individuals. The major G-allele of LYPLAL1 rs2605100 associated with increased fasting serum triglyceride concentrations  = 3%), padditive = 2.7×10−3), an association driven by the male gender (pinteraction = 0.02). The same allele associated with increased fasting serum insulin concentrations , padditive = 2.5×10−3) and increased insulin resistance (HOMA-IR) , padditive = 1.5×10−3). The minor G-allele of rs10146997 in NRXN3 associated with increased WC among women , padditive = 1.7×10−3, pinteraction = 1.0×10−3), but showed no associations with obesity related metabolic traits. The MSRA rs545854 and TFAP2B rs987237 showed nominal associations with central obesity; however, no underlying metabolic phenotypes became obvious, when investigating quantitative metabolic traits. None of the variants influenced the prevalence of type 2 diabetes.The four variants were genotyped in Danish individuals using KASPar®. Quantitative metabolic traits were examined in a population-based sample LYPLAL1, NRXN3, MSRA, and TFAP2B associate with metabolic and anthropometric traits in Danish adults. However, analyses were made without adjusting for multiple testing, and further studies are needed to confirm the putative role of LYPLAL1, NRXN3, MSRA, and TFAP2B in the pathophysiology of obesity.We demonstrate that several of the central obesity-associated variants in  To provide new insight into the pathophysiology of obesity, genome-wide association studies (GWAS) have in the last few years been performed with elaborating results LYPLAL1, is thought to act as a triglyceride lipase and reported to be up-regulated in subcutaneous adipose tissue of obese individuals LYPLAL1 associated with increased WHR (p = 2.6×10−8) only among women NRXN3 encodes the neuroxines protein family that functions in the vertebrate nervous system as cell adhesion molecules and receptors. In patients with schizophrenia, NRXN3 has been demonstrated to associate with alcohol dependence and the degree of nicotine dependence NRXN3 associated with WC (p = 6.4×10−7) and BMI (p = 7.4×10−6) in the CHARGE GWAS; however, the association with WC attenuated (p = 0.32), when adjusted for BMI MSRA encodes an antioxidant enzyme that repairs proteins inactivated by oxidative damage, but the biological connections between the MSRA locus and adiposity are unclear MSRA and central obesity measured by WC (p = 8.9×10−9)  p = 0.11), when adjusted for BMI.Lysophospholipase-like 1 protein, encoded by TFAP2B is expressed in adipose tissue TFAP2B and type 2 diabetes, and over-expression of TFAP2B in adipocytes leads to IR and accumulation of triglycerides inside the adipocytes et al. found that men carrying the 4 repeat allele of intron 2 polymorphism (intronic variable number tandem repeat) of TFAP2B had higher insulin sensitivity and central obesity (measured by skin folds) TFAP2B might be a novel candidate gene for development of the metabolic syndrome, as it was shown to regulate the expression of various adipokines TFAP2B associated significantly with central obesity measured by WC (p = 1.9×10−11) and general obesity measured by BMI ) in the mixed-gender analysis The binding-protein encoded by LYPLAL1, NRXN3, MSRA, and TFAP2B for associations with quantitative metabolic traits in a population-based sample of 6,038 adult Danes (The Inter99 study sample). In addition, we examined associations between these variants and anthropometric measures  in a larger group of individuals. Also, we conducted large case-control studies involving a total of 15,326 Danes, investigating associations between these variants and type 2 diabetes, as well as both central and general overweight and obesity. Since three of the variants  in previous studies have been investigated using sex-stratified analyses, we performed all analyses stratified according to sex.The aim of the present study was to investigate central obesity-associated variants in LYPLAL1 associated with fasting serum triglyceride concentrations  = 3%, )1;5), padditive (add) = 2.7×10−3), fasting serum insulin concentrations , padd = 2.5×10−3), and homeostasis model assessment-insulin resistance (HOMA-IR) , padd = 1.5×10−3) (padd = 2.4×10−4) (intp = 0.02) , padd = 0.05), and decreased WC , padd = 0.04) (padd = 0.47) (padd = 0.004) and BMI , padd = 0.01), but the associations diminished, when data were adjusted for BMI (padd = 0.12) and WC (padd = 0.87), respectively (add = 0.92 (0.86–0.98(95% CI)), padd = 0.01) (padd = 0.004)  . When st.4×10−4) . Indeed, = 0.02) . Further = 0.04) . However = 0.47) . The comectively . In the  = 0.01) , and the= 0.004) . No othe= 0.004) .NRXN3 associated with increased WC among women , padd = 1.7×10−3) (pint = 1.0×10−3) (add = 1.02 (0.94–1.11), padd = 0.66)   in the I.0×10−3) . The sig.0×10−3) . This as.0×10−3) . Neither = 0.66) . No inteMSRA associated with decreased fasting serum insulin concentrations , padd = 0.02), and HOMA-IR , padd = 0.02) among men (intp = 0.08) (add = 1.08 (1.00–1.18), padd = 0.05) (padd = 0.02)  . The var = 0.08) , and S6. = 0.05) , and whe = 0.02) .TFAP2B did not associate with any metabolic traits in the QT analysis (add = 1.08 (1.0–1.17), padd = 0.06) in the case-control analysis (add = 1.17 (1.03–1.32), padd = 0.01; pint = 0.04). When adjusted for BMI, the association became stronger among women (padd = 0.001) and it also became evident among men (padd = 0.01)  .TFAP2B rs987237 in the combined QT analysis in relation to general obesity (BMI)   .None of the variants associated with central and general overweight, or type 2 diabetes in the case-control analyses (data not shown).LYPLAL1, NRXN3, MSRA, and TFAP2B.In this study we found several associations with quantitative metabolic and anthropometric traits for the central obesity-associated variants in LYPLAL1 associated with elevated concentrations of fasting serum triglycerides and fasting serum insulin and with estimates of central obesity. In a recent meta-analysis of 32 GWAS for WHR, a variant in LYPLAL1 (rs4846567) in high linkage disequilibrium  with rs2605100 achieved genome-wide significance, and the subsequent association analyses with related metabolic traits in 77,167 participants  showed that this variant associated with increased levels of serum triglycerides (p = 0.018), fasting insulin (p = 1.1×10−5), and HOMA-IR (p = 9.8×10−6) As a major finding the G-allele  of rs2605100 in LYPLAL1 are in line with previous observations that a higher fat-mass causes increased lipogenesis, resulting in higher levels of circulating triglycerides and IR LYPLAL1 variant and obesity.Our findings for rs2605100 in NRXN3 associated with increased WC among women, but we found no clear association with BMI as reported in a recent meta-analysis of 249,796 individuals NRXN3  in the meta-analysis The minor G-allele rs10146997 in MSRA rs545854 minor G-allele showed associations with decreased fasting levels of serum insulin and decreased HOMA-IR among men, but no association with obesity was evident. The latter finding is consistent with the recent meta-analyses that failed to demonstrate genome-wide significant evidence of associations between this variant and BMI or WHR TFAP2B showed borderline association with central obesity among women. The GWAS showed no evidence of sex-specific associations. However, TFAP2B was confirmed as an obesity locus in the recent meta-analysis, where this variant associated with BMI at a genome-wide significant level In this study, the It is well known that substantial gender-specific differences in fat distribution exist NRXN3 is expressed in the brain, as many of the other recently discovered obesity loci, and variants in NRXN3 may provide changes of the brain function and behavior NRXN3 has been investigated in relation to addiction of alcohol and smoking MSRA is thought to be involved in the oxidative damage in cells but the molecular mechanisms are not yet elucidated. Another candidate gene located near MSRA, the TRF1-interacting ankyrin-related ADP-ribose polymerase (TNKS), has been proposed as the causal gene for the association with central obesity TFAP2B has been investigated for associations with several phenotypes correlated to diabetes and obesity TFAP2B minor G-allele, however, we were not able to determine the underlying phenotype for this association in the quantitative trait analysis of obesity-related measures, nor did we show any association with type 2 diabetes.Whether variation in the examined four genes is causal in relation to obesity remain unclear. LYPLAL1, NRXN3, MSRA, and TFAP2B associated with metabolic and anthropometric traits among adult Danes. However, analyses were made without adjusting for multiple testing, and further studies are needed to elucidate the involvement of LYPLAL1, NRXN3, MSRA, and TFAP2B in the pathophysiology of obesity.In conclusion, we found that several of the central obesity-associated variants in The studies were approved by the appropriate Regional Ethics Committees, and were in accordance with the principles of the Helsinki Declaration.n = 6,162 individuals, aged 30–61 years) from the Research Centre for Prevention and Health in Glostrup, Copenhagen  n = 1,695) sampled at Steno Diabetes Center (SDC), 3) a randomized, population-based group of unrelated middle-aged individuals (n = 730) examined at SDC, Copenhagen, and 4) The Danish ADDITION screening cohort , which is part of the Anglo-Danish-Dutch study of Intensive Treatment in People with Screen-detected Diabetes in Primary Care  This study comprises 15,326 Danish individuals ascertained from four different study groups : 1) the n = 124) were excluded from the QT analyses.A standandardized oral glucose tolerance test was undertaken in volunteers of Inter99. In this cohort QT analyses were performed in treatment-naive individuals involving individuals with normal glucose tolerance, impaired fasting glucose and impaired glucose tolerance. The exact numbers are given in tables. Patients with known type 2 diabetes  in MSRA, and rs987237 in TFAP2B were genotyped using KASPar® with success rates >97% and error rates  =  0%. The risk-allele frequencies were 70% , 21% , 15% , and 17% , which are in accordance with HapMap, and obeyed Hardy-Weinberg equilibrium (p>0.05).The rs2605100 in P-values given in parentheses were only adjusted for age and sex. Values of serum triglyceride, serum insulin, HOMA-IR, and plasma glucose had non-normally distributed residuals and were logarithmically transformed prior to statistical analyses. Their effect sizes (β) are presented as an increase/decrease in percent with 95% CI. Values without transformation  are given as actual values with 95% CI. The case-control studies were analyzed using logistic regression, assuming an additive model. Here we included type 2 diabetes patients, why these analyses were adjusted for sex, age and diabetes treatment, and with or without BMI or WC, respectively. Heterogeneity was assessed with the generic inverse variance meta-analysis method (R package: meta), which describes the proportion of variation in the effects that is attributable to genuine differences across the study groups rather than to random error.The four variants were investigated for associations with quantitative metabolic traits. The quantitative variables were tested for differences between genotyped groups using linear regression, assuming an additive model. All analyses were adjusted for sex, age, and BMI, when appropriate. BMI was additionally adjusted for WC. P-values were not adjusted for multiple hypothesis testing and p<0.05 was considered nominally significant.To investigate whether the effect of the alleles differed between genders, we included an interaction term between sex and the genotype of interest in the linear model. In this model, we assumed an additive effect for the genotypes and sex as a binary vector. All statistical analyses were performed using R version 2.9.2. http://www.sph.umich.edu/csg/abecasis/cats/. Depending on the type of analysis, as well as the risk allele frequency (RAF) of the analyzed variant, we had between 72% and 100% power to detect genetic effects in this study (p<0.05)  . The low(p<0.05) . The sta of 0.05 .Table S1Characteristics for individuals included in the analyses stratified according to study group. Data are means ± standard deviation. SDC, Steno diabetes center. WC, waist circumference.(DOCX)Click here for additional data file.Table S2Quantitative metabolic traits in 5,769 treatment-naïve middle-aged Danes from the population-based Inter99 cohort according to LYPLAL1 rs2605100 genotype. Data are unadjusted means ± standard deviation or medians (interquartile range). Values of fasting serum triglycerides, serum insulin, plasma glucose, and HOMA-IR were logarithmically transformed prior to statistical analyses, and their effect sizes (β) are presented as the increase/decrease in percent. P-values were calculated assuming an additive model (padd). Interaction analysis (pint) was performed to test whether the effect of the alleles differed between men and women. All analyses were adjusted for age, sex, and BMI. Furthermore, waist circumference and waist-hip ratio were adjusted for age and sex, and the p-values are shown in parentheses.(DOCX)Click here for additional data file.Table S3LYPLAL1 rs2605100, NRXN3 rs10146997, MSRA rs545854, and TFAP2B rs987237 in relation to central obesity. Data are number of individuals, divided into genotype groups. The effect is either the odds ratio (OR) or the per allele effect size presented as the increase/decrease and 95%CI. Effect and p-values shown are for an additive genetic model (padd) and are adjusted for age, sex and diabetes treatment (without/ with BMI) for the obese cases, and QT analyses are adjusted for age and sex (without/with BMI). QT, quantitative trait; WC, waist circumference; WHR, waist-hip ratio.(DOCX)Click here for additional data file.Table S4LYPLAL1 rs2605100, NRXN3 rs10146997, MSRA rs545854, and TFAP2B rs987237 in relation to general obesity. Data are number of individuals, divided into genotype groups. The effect is either the odds ratio (OR) or the per allele effect size presented as the increase/decrease and 95%CI. Effect and p-values shown are for an additive genetic model (padd) and are adjusted for age, sex and diabetes treatment (without/ with WC) for the obese cases, and QT analyses are adjusted for age and sex (without/with WC). QT, quantitative trait.(DOCX)Click here for additional data file.Table S5Quantitative metabolic traits in 5,789 treatment-naïve middle-aged Danes from the population-based Inter99 cohort according to NRXN3 rs10146997 genotype. Data are unadjusted means ± standard deviation or medians (interquartile range). Values of fasting serum triglycerides, serum insulin, plasma glucose, and HOMA-IR were logarithmically transformed prior to statistical analyses, and their effect sizes (β) are presented as the increase/decrease in percent. P-values were calculated assuming an additive model (padd). Interaction analysis (pint) was performed to test whether the effect of the alleles differed between men and women. All analyses were adjusted for age, sex, and BMI. Furthermore, waist circumference and waist-hip ratio were adjusted for age and sex, and the p-values are shown in parentheses.(DOCX)Click here for additional data file.Table S6Quantitative metabolic traits in 5,804 treatment-naïve middle-aged Danes from the population-based Inter99 cohort according to MSRA rs545854 genotype. Data are unadjusted means ± standard deviation or medians (interquartile range). Values of fasting serum triglycerides, serum insulin, plasma glucose, and HOMA-IR were logarithmically transformed prior to statistical analyses, and their effect sizes (β) are presented as the increase/decrease in percent. P-values were calculated assuming an additive model (padd). Interaction analysis (pint) was performed to test whether the effect of the alleles differed between men and women. All analyses were adjusted for age, sex, and BMI. Furthermore, waist circumference and waist-hip ratio were adjusted for age and sex, and the p-values are shown in parentheses.(DOCX)Click here for additional data file.Table S7Quantitative metabolic traits in 5,764 treatment-naïve middle-aged Danes from the population-based Inter99 cohort according to TFAP2B rs987237 genotype. Data are unadjusted means ± standard deviation or medians (interquartile range). Values of fasting serum triglycerides, serum insulin, plasma glucose, and HOMA-IR were logarithmically transformed prior to statistical analyses, and their effect sizes (β) are presented as the increase/decrease in percent. P-values were calculated assuming an additive model (padd). Interaction analysis (pint) was performed to test whether the effect of the alleles differed between men and women. All analyses were adjusted for age, sex, and BMI. Furthermore, waist circumference and waist-hip ratio were adjusted for age and sex, and the p-values are shown in parentheses.(DOCX)Click here for additional data file.Table S8Statistical power estimates.Central overweight: ∼4,500 controls and ∼3,700 overweight individuals. Prevalence = 0.23 (Men with waist circumference ≥94 cm and <102 cm and women with waist circumference ≥80 cm and <88 cm from the Inter99). General overweight: ∼3,200 controls and ∼7,000 overweight individuals. Prevalence = 0.39 . Central obesity: ∼4,500 controls and ∼7,000 obese individuals. Prevalence = 0.22 (men with waist circumference ≥102 cm and women with waist circumference ≥88cm from the Inter99). General obesity: ∼3,200 controls and ∼4,800 obese individuals. Prevalence = 0.17 . Type 2 diabetes: ∼ 4,900 controls and ∼ 3,500 type 2 diabetics. Prevalence = 0.05 . The statistical power calculations for quantitative traits were estimated in R using 1,000 simulations and a significance threshold of 0.05. The statistical power calculations in the case-control analyses were done using CaTS, power calculations for large genetic association studies, available at http://www.sph.umich.edu/csg/abecasis/cats/. RAF  =  risk allele frequency.(DOCX)Click here for additional data file.


EA  was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.The present study was designed to evaluate different doses of ellagic acid (EA)  Ellagic acid (EA) is one of the important components of fruits and vegetables  and has Aromatase represents a crucial enzyme of estrogen biosynthesis, and increased expression of aromatase has been observed in breast cancer tissue . FlavonoAnother possible mechanism for the broad chemoprotective and antioxidant effects of EA might involve the induction of phase II and antioxidant enzymes. GSTs have a considerably important role in the detoxification of carcinogens . NQO1 prin vivo treatment with different dose of EA by investigating changes in the activity, mRNA, and protein levels of specific rat hepatic P450s, phase II, and antioxidant enzymes at once.Although there are individual studies examining the effect of EA on several enzymes, no study has evaluated the alteration of so many enzymes simultaneously with different dose of EA. Therefore, the present study was undertaken to observe the overall simultaneous changes following β-NADPH, phenol, caffeine, TRIS, PMSF, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dodecyl sulfate (SDS), and sodium potassium tartrate. Anti-rat CYP1A1, CYP2B, CYP2C6, CYP2E1, CYP3A1, and CYP19 antibodies were from Abcam . All other chemicals and solvents were obtained from commercial sources at the highest grade of purity available.The following chemicals were purchased from Sigma-Aldrich Chemical Company : ellagic acid, acrylamide, aniline, anti-rabbit IgG-HRP conjugate, bovine serum albumin (BSA), Folin phenol reagent, glycerol, glycine, HEPES, Healthy male Wistar rats, about 12 weeks old and weighing 200–250 g, were obtained from the University Animal House. They were housed in small cages at an ambient temperature of 22 ± 1°C, on a 12 h light/dark cycle, and a standard pellet diet and distilled water were available without restriction. All experimental procedures with the animals were performed under appropriate regimes with veterinary services within the licensed projects (PAUHADEK-2009-007).After being acclimatized for 1 week, the rats were randomized and divided into three groups. Ellagic acid was given intragastrically to two experimental groups (15 rats per group) in a dose of 10 mg/kg/day (T10) and 30 mg/kg/day (T30) dissolved in DMSO, respectively. Control (15 rats) rats only received a vehicle (DMSO). The doses used were chosen to be similar to the doses used in previous studies in the literature –26. The ε-aminocaproic acid, 0.15 mM butylated hydroxytoluene, and 0.025% Triton X-100) using a tissue homogenizer with a Teflon pestle at 4°C. Subcellular fractions  of rat tissues were prepared by standard differential centrifugation with calcium aggregation as described by [Tissues were homogenized in a 4 part homogenization solution , benzyloxyresorufin O-demethylase (BROD), pentoxyresorufin O-demethylase (PROD) activities were assayed as described by [S-transferase (GST) activities were assayed as described by [Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined with an autoanalyzer using audit diagnostics AST and ALT. The microsomal cytochrome P450-dependent aniline 4-hydroxylase (A4H) activities of rat microsomes were determined by measuring the quantity of p-aminophenol formed, as described by . Aminopyribed by . Dibenzyribed by . Rat livribed by . Glutathribed by . Catalasribed by , 36, resμg protein samples were separated on 8.5% polyacrylamide gels using the discontinuous buffer system of [SDS-PAGE and western blotting were performed as described previously . Brieflyystem of . Proteinμg of RNA was incubated at 70°C for 10 minutes with 0.5 μg of oligo (dT). After 5 min on ice, 50 U Moloney murine leukemia virus reverses transcriptase, 1 mM dNTPs and 5X reaction buffer were added to the previous mixture and incubated at 42°C for 60 min. The reaction was stopped by heating the mixture to 70°C for 10 min, and the cDNA was stored at −80°C for further use.Total RNA was extracted from 100 mg rat livers using TRIzol reagent. Extracted RNA was quantified spectrophotometrically at 260/280 nm, and the integrity was checked using 1% agarose gel. For cDNA synthesis, 2.5 β-actin were used as a housekeeping gene. The PCR amplification was done using Power SYBR Green PCR master mix  and 500 nmol/L of forward and reverse primers for each gene, for which the final primer concentration was 125 nmol/L each. Quantitative PCR was done using a Light Cycler 1.5 Instrument . The PCR conditions were as follows: DNA polymerase activation at 95°C for 15 minutes, followed by 45 cycles at 95°C for 10 seconds, 54–57°C annealing (depending on the gene) for 5 seconds, and 72°C for 30 seconds. All gene analyses were done at least three times. qRT-PCR assay was performed by using gene specific primers. The oligosequences used as forward and reverse primers for rat CYP450 isozymes were based on those reported in , 39. Glyt-test, and P < 0.05 was selected as the level required for statistical significance. Statistical comparisons between three groups were assessed by one-way analysis of variance (ANOVA). When F ratios were significant (P < 0.05), one-way ANOVA was followed by Tukey's Post hoc test for comparisons of multiple group means.Statistical analyses were performed using the Minitab 13 statistical software package . All results were expressed as means including their Standard Error of Means (SEMs). Comparison between groups was performed using Student's Control and treated rats showed no significant differences in food consumption or body weight, (data not shown) after intragastric delivery of the EA to the animals at two different doses (T10 and T30).As shown in P < 0.05) 13%, 10%, 15%, and 10% or 55%, 38%, 23%, and 30% decrease in these activities in the liver, respectively. The effect of the EA on CYP2B-associated BPND, EmND, BROD, and PROD activities is presented in CYP2E-associated A4H and NDMA activities in control and EA-treated rats are given in The effect of the EA on CYP2C6-associated APND, CYP3A-associated ERND, and CYP19-associated DBFOD activity is presented in P < 0.05) in the T30 treated group. Furthermore, treatment of rats with T30 dose caused 194%, 64%, and 114% increases in NQO1, CAT, and GPX activities, respectively, when compared to the control values  was not effective because its concentration might not have been enough to reach to the site of action and quench all free radicals generated, the higher dose of EA (30 mg/kg/day) was good enough to be effective. In conclusion, the dose-dependent suppression of CYP1A, CYP2E, and CYP19 and the induction of GSTs, NQO1, GPX, and CAT enzymes suggest anticancer as well as chemopreventive roles for EA while reductions in CYP2B, 2C, and 3A explain potential drug interactions.