Epigenetic mechanisms, including histone acetylation and DNA methylation, have been widely implicated in hippocampal-dependent learning paradigms. Here, we have examined the role of epigenetic alterations in amygdala-dependent auditory Pavlovian fear conditioning and associated synaptic plasticity in the lateral nucleus of the amygdala (LA) in the rat. Using Western blotting, we first show that auditory fear conditioning is associated with an increase in histone H3 acetylation and DNMT3A expression in the LA, and that training-related alterations in histone acetylation and DNMT3A expression in the LA are downstream of ERK/MAPK signaling. Next, we show that intra-LA infusion of the histone deacetylase (HDAC) inhibitor TSA increases H3 acetylation and enhances fear memory consolidation; that is, long-term memory (LTM) is enhanced, while short-term memory (STM) is unaffected. Conversely, intra-LA infusion of the DNA methyltransferase (DNMT) inhibitor 5-AZA impairs fear memory consolidation. Further, intra-LA infusion of 5-AZA was observed to impair training-related increases in H3 acetylation, and pre-treatment with TSA was observed to rescue the memory consolidation deficit induced by 5-AZA. In our final series of experiments, we show that bath application of either 5-AZA or TSA to amygdala slices results in significant impairment or enhancement, respectively, of long-term potentiation (LTP) at both thalamic and cortical inputs to the LA. Further, the deficit in LTP following treatment with 5-AZA was observed to be rescued at both inputs by co-application of TSA. Collectively, these findings provide strong support that histone acetylation and DNA methylation work in concert to regulate memory consolidation of auditory fear conditioning and associated synaptic plasticity in the LA. Traditional views of memory formation have emphasized the importance of NMDA receptor (NMDAR)-driven alterations in protein kinase signaling cascades, the activation of transcription factors, and associated changes in gene expression that are thought to be critical for long-term memory and synaptic plasticity Within the last decade, it has become increasingly clear that epigenetic mechanisms, including modifications of chromatin structure and DNA methylation, play an additional critical role in transcriptional regulation, synaptic plasticity, and memory formation While studies have pointed to a clear and vital role for epigenetic alterations in hippocampal-dependent memory formation, few studies have systematically examined the role of epigenetic mechanisms in amygdala-dependent memory formation Epigenetic mechanisms, including histone acetylation and DNA methylation, have been widely implicated in memory formation, primarily hippocampal-dependent memory tasks such as contextual fear conditioning and object recognition In our first series of experiments, we examined changes in histone acetylation and DNMT expression in the LA following auditory fear conditioning using Western blotting . Rats we = 4.73, p<0.01], while levels of histone H4 acetylation remained unchanged  or H4  at any time point   = 1.06, p>0.05] suggesting an equal amount of protein was loaded within each group.Representative Western blots can be viewed in me point , indicat  = 3.45, p<0.01], while levels of DNMT3B remained unchanged .Analysis of DNMT protein expression revealed a significant increase in DNMT3A , with H3 acetylation in the Paired group differing significantly from that of Tone Alone, Imm Shock, and Naïve groups . No differences were observed between any of the other groups (p>0.05). Further, total levels of histone H3  did not differ between the groups.In our next series of experiments, we used Western blotting to ask whether the increase in H3 acetylation and DNMT3A expression in the LA at 90 min after fear conditioning is the result of the tone-shock pairing rather than to presentation of either tone or shock alone. Rats were presented with either 3 tone-shock pairings (“Paired”), 3 tones in the absence of shock (“Tone Alone”), or 3 immediate shocks of equivalent duration and intensity to that of the Paired group (“Imm Shock”). Ninety min later, rats in each group were sacrificed and punches were taken from the LA . A naïve  = 3.25, p<0.05], with DNMT3A expression in the Paired group differing significantly from that of Tone Alone, Imm Shock, and Naïve groups . No differences were observed between any of the other groups (p>0.05). No significant regulation of DNMT3B expression was observed . Further, levels of the loading control GAPDH did not differ between the groups .Western blot analysis of DNMT3A expression can be viewed in (13)  = 2.37, p<0.04] in the LA, while levels of H4 acetylation remained unchanged  and H4  remained unchanged following U0126 infusion ((14)  = 1.19, p>0.05]. Similarly, treatment with U0126 was observed to significantly impair the training-related increase in DNMT3A expression in the LA , while levels of the loading control GAPDH did not differ between groups .We next asked whether the training-related regulation of histone H3 and DNMT3A expression in the LA is downstream of ERK/MAPK signaling. Previous studies have shown that auditory fear conditioning regulates ERK1/2 activation in the LA, with peaks at both 5 and 60 min following training infusion , and theThus, amygdala-dependent auditory Pavlovian fear conditioning regulates histone H3 acetylation and DNMT3A expression in the LA in an ERK-dependent manner. Further, this training-related increase in H3 acetylation and DNMT3A expression is the result of the associative pairing of tone and shock rather than to presentation of either stimulus alone.In our initial series of experiments we showed that auditory fear conditioning regulates H3 acetylation. In our next series of experiments, we asked whether this training-related increase in H3 acetylation is required for the formation and/or consolidation of auditory fear conditioning. Several recent studies have indicated that intra-CA1 infusion of HDAC inhibitors increases H3 acetylation in the hippocampus and enhances consolidation of a contextual fear memory (14)  = 2.44, p<0.03] and H4  acetylation in TSA-infused rats relative to vehicle-infused controls ((14)  = 0.45, p>0.05] or H4  did not differ between the two groups ((14)  = 0.13, p>0.05; H4: t(14)  = 0.29, p>0.05; not shown]. Representative blots can be viewed in In our first experiment, we determined whether intra-LA infusion of TSA results in enhanced histone acetylation in the LA. Rats were trained with 2 tone-shock pairings followed 1 hr later by intra-LA infusion of vehicle or TSA (1 µg/side). This slightly weaker training protocol  was used to avoid a ceiling effect in freezing which may have prevented our ability to detect a memory enhancing effect of TSA. Thirty min following infusion (90 min after training) rats were sacrificed and punches were taken from the LA . Westerncontrols . Importao groups . Further  = 1.52, p>0.05] and the group by trial interaction , but a significant effect for trial . During the LTM test, however, TSA-infused rats demonstrated a higher level of fear memory retention relative to vehicle-infused controls   = 15.86, p<0.002] and trial , but a nonsignificant group by trial interaction . Cannula placements are depicted in To examine the effect of intra-LA HDAC inhibition on fear memory consolidation, rats were trained with 2 tone-shock pairings as above followed 1 hr later by intra-LA infusion of either vehicle or TSA   = 0.01, p>0.05] or the group by trial interaction ; however there was a significant main effect for trial . Further, TSA and vehicle-infused rats exhibited comparable levels of freezing during the STM test   = 0.01, p>0.05], trial , and the group by trial interaction . Therefore, TSA does not interfere with acquisition or STM formation of an auditory fear memory in the LA. Cannula placements for rats infused with either vehicle or TSA are depicted in Next, we examined the effects of intra-LA infusion of TSA on STM in a separate group of rats. Rats were trained and infused as above and STM was assessed in a distinct context 1 hr following infusion . No diffSTM test . The ANODNA methylation has been shown to negatively regulate memory via its ability to repress gene transcription   = 0.22, p>0.05] or the group by trial interaction ; however, there was a significant main effect of trial . The results of the LTM test are depicted in   = 22.15, p<0.001] and trial , but a nonsignificant group by trial interaction . Cannula placements for rats in this experiment are depicted in Rats were trained using 3 tone-shock pairings followed 1 hr later by intra-LA infusion of vehicle or 5-AZA (1 µg/side). Long-term memory was tested 24 hrs later in a distinct context . No diff  = 3.03, p>0.05] and the group by trial interaction , but there was a significant effect for trial . Further, when long-term memory was assessed ∼24 hrs later, no significant differences were observed   = 1.00, p>0.05], trial , or the group by trial interaction , indicating that rats in the 5-AZA-infused group were in fact still able to learn at levels equivalent to that of the vehicle-infused group.To ensure that 5-AZA did not have non-specific effects or cause damage to the amygdala, we retrained and retested the groups that received vehicle or 5-AZA drug-free one week later. As before, we observed no significant differences in post-shock freezing (not shown). The ANOVA for PSF scores in the re-trained groups revealed nonsignificant effects for group  or the group by trial interaction ; however there was a significant main effect for trial . Further, freezing between 5-AZA and vehicle-infused rats was comparable during the STM test   = 0.03, p>0.05], trial , and the group by trial interaction . Therefore, 5-AZA does not interfere with acquisition or STM formation of an auditory fear memory in the LA. Cannula placements for the STM experiment are shown in Next, we examined the effect of intra-LA infusion of 5-AZA on STM. A separate group of rats was trained and infused as above, and STM was assessed in a distinct context 1 hr following infusion   = 2.54, p<0.02], but not H4  acetylation in the 5-AZA-infused group ((12)  = 0.16, p>0.05] or H4  did not differ between the two groups ((13)  = 0.40, p>0.05; H4: t(13)  = 0.78, p>0.05; not shown].In our first experiment, rats were trained with 3 tone-shock pairings followed 1 hr later by intra-LA infusion of vehicle or 5-AZA (1 µg/side). Thirty min following infusion (90 min following training), rats were sacrificed and punches were taken from the LA . Westerned group . Represeo groups . AdditioF  = 71.02, p<0.01], but no significant main effect of group  or group by trial interaction . Further, on the next day, the group infused with vehicle/5-AZA exhibited impaired LTM, while the TSA/vehicle and vehicle/vehicle infused groups showed equivalent levels of intact LTM   = 8.20, p<0.01] and trial , but no significant group by trial interaction . Thus, these findings both confirm that intra-LA infusion of a DNMT inhibitor interferes with LTM formation, and also indicate that pre-treatment with an HDAC inhibitor can rescue the consolidation deficit induced by a DNMT inhibitor. Cannula placements are shown in In our second experiment, rats were trained with three tone-shock pairings and immediately infused with either TSA (1 µg/0.5 µl) or vehicle (0.5 µl/side) followed 1 hr later by infusion of either 5-AZA (1 µg/0.5 µl) or vehicle (0.5 µl/side). All rats were given a LTM test 24 hrs later . There wtact LTM . The ANOIn our final series of experiments, we examined whether histone acetylation and DNA methylation are involved in synaptic plasticity in the LA. We first examined the effects of DNMT and HDAC inhibition on LTP at thalamic and cortical inputs to the LA. Next, we examined whether co-application of an HDAC inhibitor can rescue the DNMT inhibitor-induced impairment in LTP in amygdala slices.in vitro. Bath application of 5-AZA (30 µM) significantly impaired LTP at both inputs ((6)  = 2.73, p<0.03]. In contrast, slices treated with 5-AZA exhibited impaired LTP, potentiating to only 88.37±5.58% which did not differ significantly from baseline  but did differ significantly from ACSF controls . In our cortical LTP experiments, control slices showed 141.80±10.52% potentiation, which differed significantly from baseline . In contrast, slices treated with 5-AZA exhibited impaired LTP, potentiating to 115.51±6.03%, which did not differ significantly from baseline  but did differ significantly from control slices . Further, bath application of 5-AZA alone had no effect on routine synaptic transmission in either pathway ((8)  = 0.63, p>0.05] or cortical  input pathways.In our first series of experiments, we examined the effects of bath application of a DNMT inhibitor on LTP at thalamic  and corth inputs . In our  pathway , insets.in vitro. For this series of experiments, we used a slightly weaker LTP induction protocol that does not induce LTP in controls (see (9)  = 0.28, p>0.05]. In contrast, slices treated with TSA exhibited LTP, potentiating to 120.57±7.44% and differing significantly from baseline  and from control slices . In our cortical LTP experiments, ACSF-infused control slices failed to exhibit LTP, potentiating to 108.53±3.92% and failing to differ from baseline . In contrast, slices treated with TSA exhibited LTP, potentiating to 126.25±6.28% and differing significantly from baseline  and from control slices . Further, bath application of TSA alone had no effect on routine synaptic transmission in either the thalamic  or cortical  input pathways   = 3.16, p<0.02]. Slices treated with a combination of the drugs (30 µM 5-AZA+2.5 µM TSA) also exhibited LTP, potentiating to 120.63±4.44% and significantly differing from baseline . However, the two groups failed to differ from one another  indicating that TSA can rescue LTP in the presence of 5-AZA co-administration. In our cortical LTP experiments, ACSF treated slices exhibited significant LTP, potentiating to 119.28±4.26% and differing significantly from baseline . Slices treated with a combination of the drugs (30 µM 5-AZA+2.5 µM TSA) also exhibited LTP, potentiating to 128.28±6.88% and significantly differing from baseline  but not from ACSF controls . Further, co-application of 5-AZA and TSA alone had no effect on routine synaptic transmission in either the thalamic  or cortical  input pathways  were housed individually in plastic cages and maintained on a 12∶12 hr light/dark cycle. Food and water were provided For behavioral experiments the histone deacetylase inhibitor Trichostatin A , the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine , and the MEK inhibitor U0126  were dissolved in 100% DMSO to yield a stock concentration of 4 µg/µl, and then diluted 1∶1 in ACSF to a final concentration of 2 µg/µl prior to infusion into the brain. For slice electrophysiology experiments 5-AZA was dissolved in DMSO to a stock concentration of 30 mM and diluted in ACSF to 30 µM prior to bath application. TSA was dissolved in DMSO to a stock concentration of 2.5 mM and diluted in ACSF to 2.5 µM prior to bath application.Guide for the Care and Use of Experimental Animals and were approved by the Yale University Institutional Animal Care and Use Committee (Protocol #2010-10801).Under a mixture of Ketamine (100 mg/kg) and Xylazine (6.0 mg/kg) anesthesia, rats were implanted bilaterally with 26-gauge stainless steel guide cannulas (Plastics One) aimed at the LA . Guide cannulas were fixed to screws in the skull using a mixture of acrylic and dental cement, and a 31-gauge dummy cannula was inserted into each guide to prevent clogging. Rats were given Buprenex (0.2 mg/kg) as an analgesic and given at least five days to recover prior to experimental procedures. All procedures were conducted in accordance to the National Institutes of Health To examine the time course of H3/H4 acetylation and DNMT 3A/3B expression following fear conditioning, rats were habituated to handling and to the conditioning chambers for four days prior to training. On the training day, rats were conditioned with 3 tone-shock pairings consisting of a 20 sec, 5 kHz, 75 dB tone that co-terminated with a 1 sec, 1 mA foot shock (ITI = 120 sec). Rats were then sacrificed using an overdose of chloral hydrate (600 mg/kg) and decapitated at either 30, 60, or 90 min following training. Naïve rats were sacrificed on the same day without training. To examine the associative regulation of histone acetylation and DNMT expression following fear conditioning, rats assigned to naïve (“Naïve”), tone alone (“Tone Alone”), or paired (“Paired”) groups were habituated to handling and to the conditioning chambers for four days prior to training. Rats assigned to the immediate shock (“Imm. Shock”) condition were handled for 4 days without exposure to the conditioning chamber to prevent context learning For Western blotting, punches were taken from the LA using a 1 mm punch tool  from 400-µm-thick sections cut on a sliding freezing microtome. Punches were manually dounced in 100 µl of ice-cold hypotonic lysis buffer . Sample buffer was immediately added to the homogenates, and the samples were boiled for 4 min. Homogenates were electrophoresed on 18% (H3/H4) or 10% (DNMT3A/B) gels and blotted to Immobilon-P . Western blots were blocked in 5% milk in TTBS buffer  then incubated with anti-acetyl-histone H3 , anti-histone H3 , anti-acetyl-histone H4 , anti-histone H4 , anti-DNMT3A , or anti-DNMT3B  antibody. Blots were then incubated with anti-rabbit conjugated to horseradish peroxidase  and developed using West Dura chemiluminescent substrate (Pierce). GAPDH  was used as a loading control for all Western blotting experiments. Optical densities of the bands were analyzed using NIH Image software. For analysis of H3/H4, optical densities for acetyl- or total histone H3/H4 were normalized to GAPDH for each sample and expressed as a percentage of that in the Naïve control group.Cannulated rats were habituated to handling, the conditioning chamber, and dummy cannula removal for 2 days prior to training. On the training day, rats were conditioned with either 2 (TSA experiment) or 3 (5-AZA experiment) tone-shock pairings consisting of a 20 sec, 5 kHz, 75 dB tone that co-terminated with a 1 sec, 0.5 mA (TSA) or 1 mA (5-AZA) foot shock (ITI = 120 sec). The weaker training protocol was used for the TSA experiment to avoid a ceiling effect, which might obscure our ability to detect enhanced memory consolidation following infusion of the HDAC inhibitor. One hour following training, rats were given intra-LA infusion of either 50% DMSO in ACSF , TSA  or 5-AZA . Infusion cannulas remained in the guides for 1 min following infusion to allow for drug diffusion from the tip. Testing for STM and LTM occurred at 2 and 24 hrs following training, respectively, in separate groups of rats. For each test, rats were placed in a distinct environment that was dark and consisted of a flat black plastic floor that had been washed with a peppermint-scented soap and were exposed to 3 conditioned stimulus (CS) tones (STM test) or 9 tones (LTM test).In a separate behavioral experiment, we examined the ability of the HDAC inhibitor TSA to rescue the consolidation deficit induced by the DNMT inhibitor 5-AZA. Rats were trained with 3 tone-shock pairings consisting of a 20 sec, 5 kHz, 75 dB tone that co-terminated with a 1 sec, 1.0 mA foot shock. Immediately after training, rats received infusions of either TSA (1 µg in 0.5 µl/side) or vehicle (0.5 µl/side), followed 1 hr later by either 5-AZA (1 µg in 0.5 µl/side) or vehicle (0.5 µl/side). Rats were then tested for LTM the next day.p<0.05.Freezing behavior, defined as a lack of all movement with the exception of that required for respiration, was recorded and expressed as a percent of the total CS presentation time. Freezing was scored using automated activity monitors (Coulbourne Instruments Model # H10-24A) mounted on the top of each behavior chamber. Data were analyzed with repeated-measures ANOVA. Differences were considered significant if To examine whether training-related elevations in histone H3 acetylation and DNMT3A expression are downstream of ERK/MAPK signaling in the LA, rats received intra-LA infusion of either 50% DMSO vehicle or the MEK inhibitor U0126  30 min prior to auditory fear conditioning consisting of 3 tone-shock pairings  and were sacrificed 90 min after training. This infusion to training interval for U0126 has previously been shown to reliably impair memory consolidation of auditory fear conditioning Three to five week old male Sprague Dawley rats were deeply anesthetized with Ketamine (100 mg/kg) and rapidly decapitated. Brains were quickly extracted and placed in a dish filled with oxygenated ice-cold ACSF. 400 µm-thick coronal sections containing the LA were cut using a Vibratome and collected in a chamber containing 32°C oxygenated ACSF for 30 min. Prior to recordings, slices were allowed to return to room temperature for at least one hour. An upright microscope equipped with infrared differential interference contrast optics (IR-DIC) was used to perform field recordings under visual guidance. Glass electrodes were filled with ACSF and had resistances of 4–8 MΩ. Stimuli (150 µsec duration) were delivered through bipolar stainless steel electrodes.For “thalamic” recording experiments, stimulation electrodes were placed in the ventral striatum, just medial to the LA. This stimulating protocol activates fibers that originate, at least in part, in the auditory thalamus http://www.cns.nyu.edu/~sanes/slice_software/) written for Igor (NIH DC00540). Field potentials were extracted from Igor and analyzed using Spike 2 software. In all experiments, the amplitude of the negative-going field potential was measured, and LTP for each time point was expressed as a percentage of the pre-induction baseline. For analysis, measurements of evoked responses during the last 10 min of the recording session were compared with the last 5 min of the baseline period using Student's t-tests.Data were acquired using Slice software (


Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT  assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α , Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway. Pifithrin-alpha (PFT-α), a p53 transcriptional inhibitor, was purchased from Sigma-Aldrich Inc. . Antibodies anti-p53, Bcl-2, Bax and β-actin antibodies were obtained from Cell Signaling Technology . HRP-conjugated secondary antibody purchased from EarthOx .LDH Cytotoxicity Assay Kit . P53 siRNA and its reference substance were purchased from Santa Cruz Biotechnology Inc. . All these agents above-mentioned were diluted to the destined concentration. DMSO was used as a solvent control.2 at 37°C. HepG2 cells were grown in standard media, when the proliferation of cell is 70%–80% cells were treated with different concentrations of DHM.Human hepatoma HepG2 cell line was offered by Affiliated Hospital of Guangdong Medical College . Cells were cultured in the RPMI 1640 medium , supplemented with 10% heat-inactivated fetal bovine serum , penicillin 100 U/ml, and streptomycin 100 U/ml, and maintained in a humidified atmosphere of 95% air +5% CO4 cells per 100 μL. Cells were seeded into a 96-well plate, which was placed in an incubator overnight to allow for attachment and recovery. Brieﬂy, cells were pretreated with 10, 25, 50, 75 and 100 μM DHM for 6, 12 and 24 h, MTT was dissolved at 5 mg/ml in warm assay medium. 20 μL of MTT solution was transferred to each well to yield a final volume 120 μL/well. Plates were incubated for 4 h at 37°C and 5% CO2. After incubation, supernatants were removed, and then 150 μL DMSO was added. The plate was placed on an orbital shaker for 5 minutes, and then the absorbance was recorded with reader  at 595 nm. The principle is that the succinate dehydrogenase of live cells mitochondrial restores exogenous MTT to water insoluble violet crystal Formazan, which deposits in cells, but the dead cells have not this feature. Data were expressed as the percentage of viable cells as follows: inhibitory rate (%)  =  [A595 (control–A595 (treated))]/[A595 (control)–A595 (blank)] ×100%.MTT assay: Cell densities were adjusted to 2×104 cells per 100 μL. Cells were seeded into a 96-well plate, which was placed in an incubator overnight to allow for attachment and recovery. Brieﬂy, cells were pretreated with 10, 50, 100 and 150 μM DHM for 24 h, and centrifuge the cells at 400 g for 5 min. Transfer 100 µl/well supernatant carefully into corresponding wells of an optically clear 96-well plate and add 100 µl Reaction Mixture to each well and incubate for up to 30 min at room temperature (Protect the plate from light). The absorbance was recorded with reader  at 490 nm. Calculation of the Percentage Cytotoxicity: Cytotoxicity (%)  =  (Test Sample – Low Control)/(High Control – Low Control) X 100 LDH assay: Cell densities were adjusted to ×106 per mL cell suspension. After treatment with DHM and PFT-α , cells were observed (100×) with inverted microscope .Cells were dispersed to prepare the 1×105 cells per well). 24 h later, the cells were treated with DHM  and incubated for 24 h. Then cells were collected, washed twice with cold D-hanks buffer solution, and resuspended in binding buffer (1×106 cells/mL). After 100 μL of HepG2 cells was transferred to a tube, 5 μL of FITC-conjugated Annexin V (AnnexinV-FITC) and 5 μL of propidium iodide (PI) were added followed by incubation for 15 min at room temperature in the dark. The cells were set as positive depending on the fluorescence intensity of Annexin V-FITC or PI. In early stage of apoptosis cells were Annexin V positive; whereas Annexin V and PI positive cells were considered in the late stage of apoptosis.Apoptotic cells were quantified using an Annexin V-FITC/PI kit  and detected by ﬂow cytometry , and analyzed by the software Modfit and CellQuest . The manual of the kit was strictly as follow 6 cells/mL on confocal dish. Cells were transfected at 80% confluence with the p53 siRNA plasmid and lipofectamine 2000TM reagent , according to the manufacturer's instructions. After 4 h, the medium was replaced by 1640 medium contained 10% FBS. The expression vector was transfected 24 h before treatment with different concentrations of DHM, cells with green spots were calculated under Leica fluorescence microscope .P53 siRNA and control siRNA were purchased from Santa Cruz Biotechnology Inc. Cells were seeded at a density of 1×10Western blotting analysis was performed using standard techniques as described previously According to the manufacturer's instruction, the total RNA was extracted with TRIzol® reagent (Invitrogen). RT-PCR was carried out by using an SYBR Premix Ex Taq (TaKaRa) with indicated primers (Table. 1), according to the instruction of LightCycler Real Time Cycler 480. Positive and negative controls were added. The PCR program: 95°C 4 min, 95°C 30 s, 60°C 40 s, 72°C 40 s, 12°C, forever, 40 cycles. After the reaction, the melt curves and amplification curves for all samples were recognized, then standard curves were made in Real-time quantitative PCR, meanwhile, as a housekeeping gene, 18S was utilized as an indicate.* P<0.05, ** P<0.01 or *** P<0.001 level. All figures shown in this article were obtained from at least three independent experiments.The data was analysised by GraphPad Prism 5. All results shown represent means ± SD from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistical differences were evaluated using the Student's t-test and considered significant at the It was shown that untreated HepG2 cells grew well with clear skeletons, whereas cells treated with DHM were distorted, some of them became round and floating. The number of normal cells reduced, and sloughed cells increased in a concentration dependent manner. Annexin V/PI double staining assay method was performed to detect cell apoptosis -A. Cell To confirm whether cell growth will be recovered after DHM withdrawal, HepG2 cells were treated with 50 μΜ DHM for 6 h thereafter replaced with fresh culture medium, and then cell growth was observed at 3, 6, 12 and 24 h after DHM withdrawal. Cells treated with 50 μΜ DHM for 6 h became round and floating, cell growth was inhibited and most HepG2 cells performed severe apoptosis -A. 24 hoIn this study, we also evaluated p53 expressions after DHM withdrawal. Cells were treated with 50 μΜ DHM for 6 h and then supernatant was replaced with fresh culture medium. DHM increased p53 expression, which was maintained even after DHM withdrawal at 3 h and 6 h. However, with the extension of incubation time, p53 protein degradation was observed at 12 h and 24 h after DHM withdrawal. Meanwhile, Bcl-2 protein expression levels reduced after DHM withdrawal at 3 h, 6 h and 12 h. 24 hours later, with p53 decreased, Bcl-2 protein up-regulated. Bax protein expression levels changed insignificantly during the process. All the results strongly indicated that DHM could significantly regulate Bcl-2 protein via p53 -B.Since DHM-trigged apoptosis is tightly associated with Bcl-2 related mitochondria-dependent apoptosis pathway, we further studied the correlation between p53 and Bcl-2 during DHM treatment. HepG2 cells were exposed to 50 μΜ of DHM for indicated period and the expression levels of p53 and Bcl-2 were evaluated. Fluorescence quantitative PCR results showed that the mRNA level expression of p53 increased and Bcl-2 reduced significantly with the increasing DHM concentration and treatment time, but the alteration of Bax was not obvious. -C, D.Meanwhile, p53 protein was up-regulated and Bcl-2 expression was reduced with the increasing DHM concentration and treatment time. After HepG2 cells were treated with 10, 25, 50, 100 and 150 μΜ of DHM for 12 h, p53 expression was up-regulated and Bcl-2 was reduced with the increase of DHM concentration. Cells were treated with 50 μΜ of DHM for 3, 6. 12, 24, and 48 h, p53 expression was up-regulated obviously and Bcl-2 reduced significantly with time-dependent manner, but the alteration of Bax expression was not obvious. However, levels of Bax/Bcl-2 proteins ratio in cells treated with DHM increased in a time- and dose-dependent manner. -A, BDHM treatment lead to cell apoptosis in HepG2 cell line -A. HowevDHM is known as an anticancer compound since it was characterized to be an apoptosis inducer TP53High-throughput sequencing of cancer genomes, interestingly, uncovered the surprising fact that only a small number of mutated, deleted or amplified genes were discovered in sporadic cancers. The most frequently mutated genes independently from the origin of the tumor are the DNA damage checkpoint tumor suppressor genes When DHM was blocked by PFT-α or by p53 siRNA, here we found that DHM inhibited Bcl-2 expression via increasing p53 which was supported by the results that DHM modulated p53 before Bcl-2 and Bcl-2 repression was attenuated dramatically. Undoubtedly, p53 has been regarded as a comprehensive and valuable target in cancer research


S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.α-Chlorohydrin is a common contaminant in food. Its ( For instance, ACH exposure rapidly causes rapid infertility in various mammalian species, including rats 2+ but insensitive to G proteins In mature spermatozoa, the nucleus is highly compacted in head and sperm are almost transcriptionally silent and translationally inactive There is increasing evidence that glucose metabolism is required for sperm PTP Capacitation is a time dependent process and PTP is recognized as a hallmark of capacitation 50) of SACH for rat sperm was about 100 mM  of rat sperm after incubation in capacitating medium in the presence or absent of SACH. A time-dependent elevation of P-PKAs (45 kDa – >220 kDa) during the capacitation was observed . When trPKA is regulated by cAMP level. As PKA activity was not directly affected by SACH , then we3−, a prime activator of sAC, could not support PTP and P-PKAs in rat sperm, but both of them were restored by the addition of dbcAMP and IBMX  have been applied to identification of PTP in human Few toxicological investigations have studied this essential factor. Our data demonstrate that SACH inhibits PTP in rat epididymal sperm. PTP in human ejaculated sperm was found sensitive to SACH as well (data now shown). Furthermore, similar results in rhesus macaque sperm had been observed when the sperm were exposed to 0.5 mM ACH in vitro 3—sAC–cAMP pathway to regulate P-PKAs As far as we know, there was no comprehensive study on identification of the target proteins of PKA in sperm, nevertheless it is speculated that PKA may module tyrosine kinases or tyrosine phosphatases activities by phosphorylating their Ser/Thr residues Clearly, blocking of glycolysis plays a pivotal role in SACH inhibition of this capacitation signal pathway. Previous studies have shown ACH affected sperm GAPDS activity or ATP levels mK) of sAC for ATP-Mg2+ has been reported at 10 mM mK, which means the velocity of sAC reaction could be sensitive to variation in ATP concentrations. Thus sAC could function as a cellular energy sensor Km of PKA is about 40 µM As the alteration of cAMP and ATP levels are essential to the inhibition of PTP, it is necessary to elucidate how the depletion of ATP led to a deficiency in cAMP. sAC may be the connecting point between these events. In mammalian sperm ATP is used as the substrate of sAC to synthesis to cAMP. But the affinity of sAC for ATP is 10-fold lower than the transmembrane adenylyl cyclase It should be noted that dbcAMP and IBMX failed to resort PTP or P-PKAs inhibited by 100 µM SACH, which implies that other factors besides cAMP depletion might be involved in SACH's action. The states of P-PKAs was balanced by the activities of PKA and Ser/Thr phosphatases, and the later play a critical role in regulation of P-PKAs during sperm capacitation In summary, our data demonstrated that SACH inhibited phosphorylation of PKA substrate and tyrosine residues through impairing energy generation from glycolysis and subsequent deficiency of cAMP level and PKA inactivation . ConsideAnimal ethics approval was obtained from Fudan University of Animal Ethics Committee. All procedures were conducted according to the National Ethics Committee for Care and Use of Laboratory Animals for Research of the Medical School.+), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), Dithiothreitol (DTT), Percoll, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tween 20, Tris base, potassium fluoride, glucose, pyruvate, lactate, glycerol, penicillin, streptomycin, glyceraldehyde-3-phosphate, sodium pyrophosphate, sodium arsenate, oxalate, oxamate, β-mercaptoethanol, aprotinin, leupeptin and anti-α-tubulin antibody were obtained from Sigma- Aldrich . N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl-cAMP (dbcAMP) were acquired from Merck . The methyl tetrazolium (MTT) assay system was purchased from Beyotime Institute of Biotechnology. Antibodies anti-phospho-tyrosine mouse mAb (P-Tyr-100) and anti-phospho-(Ser/Thr) PKA substrate were purchased from Cell Signaling Technology .Bovine serum albumin fraction V (BSA), Nicotinamide adenine dinucleotide , and maintained five per cage in a condition with constant temperature (22±2°C) and humidity (50%), and light/dark cycle (12 hours each).4, 1.2 mM NaH2PO4, 5.6 mM glucose, 0.25 mM sodium pyruvate, 21.6 mM sodium lactate, 1.7 mM CaCl2, 25.1 mM NaHCO3, 10 mM HEPES (pH 7.5), and 15 mg/ml of BSA. BWW free of bicarbonate (Non-capacitation medium) was prepared by substituting NaHCO3 with 25.1 mM NaCl to maintain osmosis.The basic medium used for capacitating rat sperm was a modified Biggers Whitten & Whittingham (BWW) medium 2 asphyxiation and cauda epididymis were minced swiftly in 1.5 ml pre-warmed BWW medium without bicarbonate. After 5 min incubation in 37°C, 5% CO2/95% air, sperm suspension was diluted equably into proper media at final concentration of 2–4×106 cell/ml and incubated in 37°C, 5% CO2/95% air for 6 hours. Especially, if the time course of PTP during capacitation was to be set up, the sperm were collected immediately when diluted into BWW medium (time = 0 hour), and then collected at the time points of 1 hour, 3 hour, 4.5 hour and 6 hour. Sperm counting were performed with a 0.1 mm depth hemacytometer.Adult male rats were sacrificed by COAt the end of incubation or treatment, a measurement of rat sperm kinematics was performed a Computer Assisted Sperm Analysis (CASA) . The following parameters were measured: motile, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), amplitude of lateral head movement (ALH), beat cross frequency (BCF) and straightness (STR). At least 200 sperm were analyzed for each measured.Briefly, sperm were incubated for 2 hours with diluted MTT solution according to the products instruction, and then Formanzan Addition of Solubilization Solution was followed by about 4 hours incubation until solid formanzan was completely resolved. Absorbance was recorded at 560 nm.6 sperm were loaded to 10% SDS-PAGE gels for electrophoresis and then transferred to PVDF membranes  at 45 volts for 3 hours. After blocking with 3% BSA in Tris buffered saline with 0.1% Tween 20 (TBST) at 4°C overnight, the membranes were incubated with first antibodies for 2 hours at room temperature at following dilutions in TBST: anti-Phospho-Tyrosine (P-Tyr-100) 1∶2000, anti-phospho-PKA substrates 1∶1000, anti-α-tubulin 1∶5000. Washing the membranes with TBST for 3 times was followed by blotting with second antibodies conjugated with horseradish peroxidase for 2 hours. Proteins were visualized using enhanced chemiluminescence detection kit . When necessary, the membranes were stripped with stripping buffer and reprobed other target proteins. Western blot intensity was quantitated with ImageJ software (http://rsb.info.nih.gov/ij/).At the end of incubation, the sperm were collected and centrifuged at 7,000×g, 4°C for 5 min, washed 3 times with cold phosphate buffered saline (PBS), and resuspended in Laemmli sample buffer without β-mercaptoethanol and boiled for 5 min. And then the samples were centrifuged at 10,000×g, 4°C for 5 min. The supernatants were removed into a new tube and added final concentration of 5% β-mercaptoethanol then boiled for 5 min. Sperm protein extracted from 1×10Rat sperm were collected and washed with cold PBS after treatment with or without SACH, discarded the supernatant by aspiration. 600 µl sonication buffer consisted of 0.3% HCAPS, 150 mM NaCl, 1 mM DTT, 10 µg/ml aprotinin and 10 µg/ml leupeptin was added and the suspension was sonicated 3 times on ice. GAPDS enzyme reaction took place in a mixture described by Welch et al. 2O to a final volume of 25 µl. The reaction was carried out for 30 min at room temperature, and stopped by 5 min's boiling. The phosphorylated peptides were separated by electrophoresis on a 0.8% agarose gel at 100 V for 15 min. Excised the negatively charged phosphorylated bands from the gel, heated at 95°C, resolved the hot agarose with 75 µl of Gel Solubilization Solution and 50 µl of glacial acetic acid, quantitated by the absorbance at 570 nm on spectrophotometry. One unit of PKA activity was defined as the number of nanomoles of phosphate transferred to a substrate per minute per 107 sperm.Sperm PKA activity was measured using a nonradioactive PKA assay kit . Rat sperm were washed twice with cold PBS and homogenized on ice in 0.5 ml PKA Extraction Buffer. After centrifugation at 14,000×g for 5 min at 4°C, the supernatant was removed. The reaction system contained 5 µl PepTag PKA reaction buffer, 5 µl PepTag A1 peptide, 5 µl cAMP, 1 µl peptide protection solution, and 4 µl sample homogenate, and added H5 rat sperm were collected and washed with cold PBS after 6 hours incubation, intracellular ATP was released by the manufacturer-supplied lysis buffer. Sample ATP extractions and a serial of ATP standard were mix with luciferase reagent and the luminescence was read immediately.ATP concentrations were analyzed using a bioluminescent ATP assay kit . In brief, 2×106 rat sperm were collected at the end of incubation, and washed with PBS by centrifuging at 5000×g, 4°C for 15 min followed by lysis and acetylation. Luminescence was read by 96 well microplate reader . Acetylation reagents, namely triethylamine and acetic anhydride, are lachrymators and should be used in hood.The DetectX cAMP Chemiluminescent Immunoassay kits  were used to measure rat sperm cAMP level. According to the instruction of the kit, 1×10The values were expressed as means ± standard error of the mean (SEM). All graphs or analyses were constructed or performed using GraphPad Prism . Comparisons between the experimental groups were analyzed using one-way ANOVA followed by Dunnett's Multiple Comparison test with the significance level set at P<0.05.Figure S1Effects of SACH on rat sperm motility. Rat sperm were incubated with 10, 25, 50 and 100 µM SACH for 6 hours in BWW, 37°C and then sperm kinematic parameters Motile, VSL, VAP, VCL, ALH, LIN, BCF and STR were measured using CASA system. Each point represents mean ±SEM, n = 3. * P<0.05 vs. control.(TIF)Click here for additional data file.Figure S2Cytotoxicity of SACH on rat sperm. Rat sperm were incubated in BWW in the presence of 0.01, 0.1, 1, 10, 100 and 1000 mM SACH for 6 hours and then a methyl tetrazolium (MTT) assay was performed. Each point presents as mean ± SEM, n = 4.(TIF)Click here for additional data file.Figure S3Effects of SACH on PKA activity in rat sperm extracts. Rat sperm were homogenized and the cytoplasm extracts were incubated with 10, 25, 50 and 100 µM SACH in PKA reaction mixture for 30 min, and then followed by PKA activity measurement. The reaction system without PKA was taken as negative control, and the positive control reaction system contained 0.4 µg/ml PKA catalytic subunit. Data represent as mean ± SEM, n = 3.(TIF)Click here for additional data file.Figure S4Glycerol restores rat sperm motility inhibited by SACH. Rat sperm were treated with 0, 0.04, 0.2 and 1.0 mM glycerol in the presence of 100 µM SACH for 6 hours in BWW, and CASA was performed to measure sperm kinematic parameters motile, VSL, VAP, VCL, ALH, LIN, BCF and STR. Data represent as mean ± SEM, n = 3.(TIF)Click here for additional data file.


Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood.2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy.We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length.We performed a dose–response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RTIn addition, we investigated BET role on myoblasts proliferation and differentiation. During proliferation, BET did not modify C2C12 proliferative rate, but promoted myogenic induction, enhancing MyoD protein content and cellular elongation. During differentiation, BET caused an increase of muscle-specific markers and IGF-1 R protein levels.Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment. Beta vulgaris) by the German chemist Scheibler, in the 19th century. Now, BET is isolated widely from microorganisms, plants and animals; it is an important component of many foods, including wheat, shellfish and spinach [Betaine (BET) is a zwitterionic quaternary ammonium compound and it was first discovered in the juice of sugar beets (S-adenosylmethionine (SAM) [BET has two main physiologic roles : 1) it ine (SAM) . Alteredne (SAM) . Many grne (SAM) ,8, inclune (SAM) , showed . First, it has been hypothesized that BET consumption improves cardiovascular function and thermo regulation while exercising in a hot environment [Some novel studies suggested a possible role of BET on enhancing exercise performanceironment . Furtherironment -14. OverMuscle remodeling is a crucial aspect of sport performance. In fact, skeletal muscle is a highly adaptable tissue that is capable not only to increase its mass in response to exercise (hypertrophy), but also to form new fibrils (regeneration) after damage -17. Bothin vitro model to study myogenesis and regulation of skeletal mass [The purpose of the present study has assess BET effects during the differentiation and hypertrophic process using C2C12 murine myoblasts. This cell line, derived from satellite cells, differentiate in myotubes after serum removal and provide a useful experimental tal mass . Our resAnti calnexin (H-70), anti MyoD (C-20), anti Myf5 (C-20), anti Myogenin (D-10), anti Myf6 (C-19), anti IGF-1 (G-17), anti MyHC (H-300), anti N-cadherin (H-63), anti α-tubulin (TU-02), anti p21 (C-19), anti IGF-1 receptor β (C-20), anti Akt (C-20), monoclonal or polyclonal primary antibodies, HRP-conjugated and rhodamine-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology . Anti α sarcomeric actinin primary antibody (A7811) and all other reagents were purchased from Sigma Chem. Co. . In particular, BET used was betaine monohydrate  and according to the manufacturer’s instruction was dissolved in sterile water. Mouse C2C12 myoblastic cells were purchased from the European Collection of Animal Cell Cultures (ECACC).2 cells/cm2, were cultured at 37°C in humidified 5% CO2 atmosphere in a growth medium (GM), containing DMEM supplemented with 20% (v/v) FBS , 1% penicillin–streptomycin and 1% L-glutamine. Cell differentiation was initiated by placing 80% confluent cell cultures in differentiation medium (DM), containing DMEM supplemented with 1% HS (horse serum), antibiotics and 1% L-glutamine [C2C12 myoblasts, seeded at 6×10lutamine .in vitro differentiation model, early myotubes appeared 48 hours (h) after serum starvation (intermediate differentiation phase) and neo myotubes formation was completed after 72 h-96 h (late differentiation phase).In our To study the dose–response relationship of BET stimulus, we treated neo myotubes with 1, 5 or 10 mM BET for 30 min, 4, 8 and 24 hours (h). Unstimulated neo myotubes represented control cells.Based on dose–response results, 10 mM BET concentration was chosen for C2C12 stimulation. To study C2C12 proliferation and differentiation, myoblasts were grown to 40-50% confluence in GM and then stimulated with BET 10 mM. Control cells were maintained in GM or DM. Medium was daily changed and cells inspected for morphological changes.2 culture dishes and grown in presence of GM supplement with 10 mM BET. Control cells were cultured in GM. Experiment continued until the control cells have reached sub-confluence (4 days). Medium was daily changed, the cells were counted using a hemocytometer and the average values for each single day were used to plot a growth curve for BET treated and control myoblasts.C2C12 myoblasts were plated in 35 mm2-PCR Array plates  to study the BET stimuli dose–response. These plates allow assessing the gene expression involved in IGF-1 pathway, glucose transport and metabolism. To analyze the BET action on cell cycle during myoblasts proliferation, we used Mouse cell cycle RT2-PCR Array plates . These plates allow to determinate the expression of 84 genes, among which genes encoding cyclins, positive and negative regulators of cell cycle, as p21 protein. In brief, total RNA was isolated from C2C12 cells using the RNeasy Plus Mini Quiagen kit  according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed using RT2-First Strand Kit . The reverse transcripts were used as templates for analysis of gene expression level, using RT2-PCR arrays plates, according to the manufacturer’s instructions. Each sample was run in triplicate. We calculated the expression level of the housekeeping genes, chosen for normalization in the threshold cycle (Ct), and then the fold-change (ΔΔCt) for each gene from treatment group compared to the control group. If the ΔΔCt is greater than 1, the result may be reported as a fold up-regulation. If the ΔΔCt is less than 1, the result may be reported as a fold down-regulation.We used Mouse Insulin RT3VO4), 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin) and shaked for 1 h at 4°C. Detergent-insoluble material was removed from the cell suspension by centrifugation at 12,000 x g for 30 min. Protein contents were quantified using Bradford method. Aliquots of 30 μg supernatant proteins from the different samples were resolved by SDS-PAGE. Electrophoresed proteins were transferred to nitrocellulose membrane  as described previously [C2C12 cells were homogenized in lysis buffer  and images were captured using IM50 software . To verify that cells number in all conditions was superimposable, nuclei were revealed with DAPI staining. To determinate myotube length dimension, the average measurement on each slide was generated from approximately 150 MyHC positive multinucleated myotubes (at least 3 nuclei). In particular, 10 fields were randomly chosen and all MyHC-positive myotubes were measured. The data were then converted to fold change (FC) of the control. Live C2C12 cells were examined and images acquired by phase contrast microscopy using the same microscope and digital system described above.Data are presented as the mean ± SD. Statistical significances were calculated using unpaired t-tests. Results were considered significant when p ≤ 0.05.in vitro. Therefore a BET dose–response experiment on neo myotubes was performed. C2C12 cells, differentiated for 72 h, were treated for additional 24 h with three different BET doses: 1 mM, 5 mM and 10 mM  with different BET concentrations  and unstimulated cells were compared , are associated with loss of skeletal muscle mass and reduced contractive force ,43,44. ISeveral authors suggested that IGF-1 pathway operates in an autocrine/paracrine mode acting as an intrinsic mediator of skeletal muscle repair and adaptation, increasing the proliferation potential of satellite cells, promoting their differentiation, enhancing muscle regeneration and eventually determining protein synthesis and increase muscle mass ,30,33,37Our data indicate BET as a positive stimulus for the activation of IGF-1 pathway in skeletal muscle. In recent years, the potential effects of BET supplementation in content of meat livestock were investigated. When added to animal feeds, BET enhanced lean muscle mass and reduced the fat . During in vitro work provides the first evidence of possible BET positive action on skeletal muscle myoblasts differentiation, in particular on the progression of the differentiation process and on myotubes morphology. This effect is at least partially mediated by the IGF-1 signaling activation. Our in vitro results are consistent with in vivo data obtained in livestock and in humans and may contribute the bench proof for a use of BET as a dietary supplement in humans.In summary, our The authors declare that they have no competing interests.PS, AM and IT designed and conducted the research and wrote the manuscript; PS, LL, AM, NM and IT analyzed the data. IT had primary responsibility for the final content. All authors read and approved the final manuscript.


Plasmodium spp. sequences from the blood of orangutans, including 1 of P. vivax, which has implications for human residents and orangutan rehabilitation programs.We identified 4 discrete  Plasmodium spp. infections in semicaptive orangutans housed at the Orangutan Care Center and Quarantine, Central Kalimantan, Indonesia, by using a combination of microscopic and DNA molecular techniques to identify the Plasmodium spp. in each animal. Previous studies indicated 2 orangutan-specific Plasmodium spp., but our data show 4 Plasmodium spp. These findings provide evidence for P. vivax transmission between humans and orangutans and for P. cynomolgi transmission between macaques and orangutans. These data have potential implications for the conservation of orangutans and also for the bidirectional transmission of parasites between orangutans and humans visiting or living in the region.Data are lacking on the specific diseases to which great apes are susceptible and the transmission dynamics and overall impact of these diseases. We examined the prevalence of  All animals also were grouped by size, which was used to estimate age: small (<15 kg), medium (15–30 kg), and large (>30 kg)  OCC&Q residents (living at OCC&Q for (>30 kg) .Blood samples were collected by an OCC&Q veterinarian with a 25-gauge × 1⅝-inch PrecisionGlide needle  and a 3-mL syringe. Some animals were sampled more than once so the effectiveness of antimalarial chemotherapy could be monitored. Thin and thick blood smears were prepared, fixed with methanol, stained with 10% Giemsa stain for 30 min, and destained with water  and rPLU Cal 2 (5´-CGC TAT TGG AGC TGG AAT TAC C-3´) in a 25-μL reaction under the conditions of 94°C (4 min) and 35 cycles at 94°C (10 sec), 60°C (10 sec), 72°C (45 sec), with an additional extension at 72°C (4 min). PCR products were size fractionated by electrophoresis on a 1% agarose gel containing ethidium bromide and examined on a BioDoc gel documentation System . A ≈500-bp band confirmed Plasmodium spp. DNA in the initial PCR sample.We used the DNA from step 1 that tested positive in step 2 in a third PCR with primers rPLU 3 (5´-TTT TTA TAA GGA TAA CTA CGG AAA AGC TGT-3´) and rPLU Cal (5´-ACA CAW RGT KCC TCT AAG AAG C-3´) by using BD Sprint Advantage Single Shots  under the conditions of 95°C (1 min), and 35 cycles at 95°C (30 sec), and 58° (3 min), with an additional extension at 58°C (3 min). These nested primers amplified an ≈1,500-bp fragment that contains 3–4 variable regions of the ribosomal sequence that can be used for species determination. After electrophoresis, PCR products were excised from the gel, purified by using a Qiagen Gel Purification Kit , and ligated into pGEM-T-Easy Vector . Putative transformants were identified by using blue-white screening of XL1-Blue Cells , grown overnight in 5 mL LB medium with ampicillin (100 μg/μL) and purified by using Wizard Plus Miniprep DNA Purification System (Promega). Sequencing of clones was done by using BigDye Chemistry (version 3.1)  and with the plasmid primers SP6 and T7 (http://www.ncbi.nlm.nih.gov/blast/). All available sequences of the Plasmodium spp. 18S sRNA gene, which contained complete sequences of our target region, were downloaded.The nucleotide sequences obtained were compared with those Plasmodium spp. sequences available in public databases by using BLASTN (nucleotide-nucleotide) , 7 from newly confiscated arrivals , and 2 newly arrived feral animals  . We aligThe 13 sequences we obtained form 4 distinct groups at the nucleotide level. On the basis of these groupings, we designated samples VS25, VS28, VM75, and VM82 as group 1; samples VS14 and VM88 as group 2; VS21, VS32, VS33, VM40, VM55, and VM71 as group 3; and VS63 as group 4. From each of these groupings, we selected 1 sequence to represent the entire group. These representative samples were then aligned at the nucleotide and translated amino acid level with available sequences downloaded from the databases to generate a phylogenetic tree .Group 1 (representative sequence VM82) consists of 1,519 bp at the nucleotide level and translates to a putative protein with a length of 506 amino acids (aa). VM82 shares the greatest sequence identity with P. cynomolgi (94%) and P. inui (95%). A phylogenetic tree shows the close association with P. inui and P. cynomolgi. We then aligned VM82 solely with its 2 closest homologs, P. inui and P. cynomolgi. These 3 sequences are highly conserved but have variable areas between bases 106–155, 633–672, 720–730, and 1,018–1,050, which indicate a closer similarity with P. cynomolgi. Because of differences between VM82 and P. cynomolgi in the region of bases 143–155, 646–673, and 1,023–1,043, we designated this group as a P. cynomolgi–like parasite.Group 2 (representative sequence VM88) consists of 1,544 bp at the nucleotide level and translates to a putative protein with a length of 514 aa. Throughout the variable regions, VM88 shares substantial similarity with P. hylobati (89%–96%), P. inui (90%–97%), P. fieldi, (89%–95%) and P. cynomolgi (90%–95%). We then aligned VM88 solely with its 2 closest homologs, P. inui and P. cynomolgi. These 3 sequences are highly conserved but show 2 large variable areas between bases 113–175 and 630–670. Despite sharing the greatest homology with P. inui, differences between VM88 and P. inui in the region of bases 110–175, 712–735, 789–887, and 1,495–1,551 indicate that this is a P. inui–like parasite.Group 3 (representative sequence VM40) consists of 1,515 bp at the nucleotide level and translates to a putative protein with a length of 505 aa. VM40 shares significant identity with 3 Plasmodium spp. at the nucleotide level: P. hylobati, (96%–97%), P. inui (95%–97%), and P. cynomolgi (96%–98%). Alignment of VM40 solely with its 2 closest sequences indicated this sequence is homologous to P. cynomolgi.Group 4, sequence VS63, consists of 1,582 bp at the nucleotide level and translates to a putative protein with a length of 526 aa. At the nucleotide level, VS63 shares the greatest identity with P. simium (97%–98%) and P. vivax (96%–98%).2 = 8.11, degrees of freedom [df] = 1, p<0.01). Because these animals were confiscated from humans, we do not know their history or often the specific geographic region from which they originated. Increased levels of stress caused by time spent in direct contact with humans, reduced arboreality, and lack of a nutritious and normal diet may contribute to increased levels of infection  or medium and large orangutans combined . Whether these results are coincidental or whether older animals have some protection against reinfections with Plasmodium spp. has not been studied. In humans, most deaths attributed to malaria occur in young children, and evidence of age-acquired immunity against Plasmodium spp. has been found  and P. vivax (found throughout Southeast Asia) and secondarily with P. knowlesi . When weP. vivax and P. simium are genetically indistinguishable at 13 microsatellite loci and 8 tandem repeats  may be infected by 4 species of plasmodia; 2 of these may represent the previously identified orangutan plasmodia P. pitheci and P. silvaticum. Macaque malaria in orangutans suggests cross-species transmission of a parasite between macaques and orangutans living sympatrically in Kalimantan  (


Hepatocellular carcinoma is the fifth most common cancer worldwide and shows a complex clinical course, poor response to pharmacological treatment, and a severe prognosis. Thus, the aim of this study was to investigate whether tacrolimus (FK506) has synergistic antitumor effects with doxorubicin on two human hepatocellular carcinoma cell lines, Huh7 and HepG2. Cell viability was analyzed by Sulforhodamine B assay and synergic effect was evaluated by the software CalcuSyn. Cell apoptosis was evaluated using Annexin V and Dead Cell assay. Apoptosis-related protein PARP-1 cleaved and autophagy-related protein expressions (Beclin-1 and LC3B) were measured by western blotting analysis. Cytokines concentration in cellular supernatants after treatments was studied by Bio-Plex assay. Interestingly the formulation with doxorubicin and tacrolimus induced higher cytotoxicity level on tumor cells than single treatment. Moreover, our results showed that the mechanisms involved were (i) a strong cell apoptosis induction, (ii) contemporaneous decrease of autophagy activation, understood as prosurvival process, and (iii) downregulation of proinflammatory cytokines. In conclusion, future studies could relate to the doxorubicin/tacrolimus combination effects in mice models bearing HCC in order to see if this formulation could be useful in HCC treatment. Streptomyces tsukubaensis that has shown antiproliferative effects in vitro [Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, and more than half a million new cases occur annually. HCC generally develops from chronic liver injury, which leads to inflammation, hepatocyte regeneration, liver matrix remodeling, fibrosis, and, finally, cirrhosis . The maiin vitro . Moreovein vitro . We havein vitro . This isin vitro , 15 and 2 in culture medium DMEM , supplemented with FBS  at 10%, penicillin/streptomycin 100× , glutamax 100× (Invitrogen), and nonessential amino acids 100× (Invitrogen). Phosphate buffer  and trypsin (Ca2+ and Mg2+ free) were supplied by Euroclone. The cells were plated 2 × 104 for well in 96-well tissue culture plates and allowed to attach for 24 h. Experiments were initiated when the cells reached 80% confluence. The cells were treated with doxorubicin  and tacrolimus (FK506)  for 48 h. The drugs were dissolved in DMEM supplemented with 1% FBS at concentrations of 0.21, 0.42, 0.85, 1.7, 3.4, and 6.8 μM and 0.013, 0.026, 0.052, 0.1, 0.2, and 0.4 μM, respectively, for Huh7 treatment, while the drugs were dissolved in DMEM supplemented with 1% FBS at concentrations of 0.09, 0.18, 0.37, 0.75, 1.5, and 3 μM for doxorubicin and 0.002, 0.006, 0.047, 0.095, 0.18, and 0.36 nM for tacrolimus during HepG2 treatment. Subsequently according to the results, combination treatment (cotreatment) with doxorubicin and tacrolimus was carried out at IC50 (the median inhibitory concentration defined as the drug concentration at which cell growth was inhibited by 50%) doses obtained for 48 h.Human hepatocellular carcinoma cell lines Huh7 and HepG2  were kept in culture and expanded at 37°C in a humidified atmosphere of 5% COAfter 48 h of drugs exposition, cell survival/proliferation was measured in presence and absence of drugs in 96-well plates by a spectrophotometric dye incorporation assay using Sulforhodamine B (SRB). Cells were fixed with trichloroacetic acid  for 1 h and after stained for 30 min with 0.4% (wt/vol) SRB (Sigma Aldrich) dissolved in 1% acetic acid. The number of viable cells was directly proportional to the protein bound-dye formation which was then solubilized with 10 mM Tris base solution pH 10.5 and measured by fluorometric assay ELISA at 540 nm . All experiments were performed in duplicate and were repeated for three times . The IC5r) of the median-effect plot is considered a conformity measure of the data according to the mass-action law principle when the experimental measurement is assumed to be accurate. An r value equal to 1 indicates perfect conformity while a poor value may be the result of biological variability or experimental deviations. For all our experiments r values were between 0.91 and 0.98 indicating a good data conformity.Drug combination studies were based on concentration-effect curves generated as a plot of the fraction of unaffected (surviving) cells versus the drug concentration after 48 h of treatment. Briefly, individually and in combination equiactive doses (50 : 50 cytotoxic ratio) of the two drugs (IC50) were tested for 48 h. Synergism, additivity, and antagonism were quantified by determining the CI (combination index) calculated by the Chou-Talalay equation and with the software CalcuSyn  . Assumin6) were harvested and washed twice with ice-cold PBS. Subsequently, the cells were labeled with Annexin V and Dead Cell assay kit according to the manufacturer's instructions . This assay is based on the phosphatidylserine (PS) detection on the apoptotic cells surface, using fluorescently labeled Annexin V in combination with the dead cell marker, 7-Aminoactinomycin D (7-AAD). We calculated the apoptotic ratio by identification of four populations: (i) viable cells, not undergoing detectable apoptosis: Annexin V (−) and dead cell marker (−), (ii) early apoptotic cells: Annexin V (+) and dead cell marker (−), (iii) late apoptotic cells: Annexin V (+) and dead cell marker (+), and (iv) cells that have died through nonapoptotic pathway: Annexin V (−) and dead cell marker (+). The samples were determined by the Muse Cell Analyzer (Merck Millipore) and analyzed by software provided by Merck Millipore.The cells  and lysed in a lysis buffer containing 50 mM N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 150 mM NaCl, 1 mM ethylenediamine tetra-acetic acid, 1 mM ethylene glycol tetra-acetic acid, 10% glycerol, 1% Triton-X-100, 1 mm phenylmethylsulfonyl fluoride, 1 lg aprotinin, 0.5 mm sodium orthovanadate, and 20 mm sodium pyrophosphate. The lysates were clarified by centrifugation at 16 000 g for 10 min. Protein concentrations were estimated by a BioRad assay (BioRad) and boiled in Laemmli buffer  4%, glycerol 20%, 2-mercaptoethanol 10%, bromophenol blue 0.002%) for 5 min before electrophoresis. Proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (15% polyacrylamide) under reducing condition. After electrophoresis, proteins were transferred to nitrocellulose membranes ; complete transfer was assessed using prestained protein standards (BioRad). After blocking with Tris-buffered saline- (TBS-) bovine serum albumin (BSA) , the membranes were incubated with the specific primary anti-human antibodies against Beclin-1 (BECN1) 1 : 500 , LC3B 1 : 500 , and PARP-1 1 : 500  overnight at 4°C. When the membranes were washed and incubated with anti-rabbit horseradish peroxidase conjugate at a dilution of 1 : 3000 for 1 h at room temperature, the immunoreactive bands of proteins were visualized by enhanced chemiluminescence immunoassay method . The blots were stripped and reprobed with anti-GAPDH antibody (Santa Cruz Biotechnology) to normalize for differences in protein loading.ββ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF basic, G-CSF, GM-CSF, INF-γ, IP-10, MCP-1, MIP-1α (CCL3), MIP-1β (CCL4), Rantes, TNF-α, and VEGF. Each experiment was performed in duplicate as previously described [A multiplex biometric ELISA-based immunoassay, the Luminex platform , containing dyed microspheres conjugated with a monoclonal antibody specific for a target protein was used, according to the manufacturer's instructions  to evaluate the trends and the concentrations of different soluble molecules in cellular supernatants. After 48 h of incubation with the drugs, used alone or in combination, some cytokines chemokines and growth factors were measured using Human Cytokine 27 Plex Panel . In particular, we evaluated PDGF-escribed , 18, 19.t-test using the statistical program Prism 4 . Values of P < 0.05 were considered to be statistically significant. Moreover, the possible interactions between the significant proteins were analyzed by Ingenuity Pathway Analysis.The cytokines concentrations evaluated in treated versus untreated Huh7 were compared by μM and 0.07 μM doses and HepG2 reached IC50 at 0.72 μM and 0.047 nM doses, respectively  and tacrolimus  induced a synergistic apoptotic effect  and LC3B. Beclin-1 is constitutively expressed in Huh7 and in HepG2 cells  and tacrolimus (0.052 μM) by Bio-Plex assay.Then, we evaluated the cytokines levels in Huh7 cellular supernatants after 48 h of incubation with drugs alone and in combination with concentrations below the IC50 values of doxorubicin  that generates networks on the basis of associated functions and data mining from experimental data reported in the literature. Interactomics studies on these cytokines were performed by IPA software. It creates networks between the selected proteins extracting information related to biological correlations from databases that collect experimental data and scientific published papers. The user can insert as input for the research only a list of genes/proteins and not other information like cell types. Our molecules have been found involved into a network named “Cell-To-Cell Signaling and Interaction, Hematological System Development and Function, Immune Cell Trafficking” .κB and its RELA subunit (NF-κB/p65), STAT3 , and ZFP36 (zinc finger protein 36) that are involved in HCC. In fact, in literature it is reported that (i) ZFP36 is responsible for inducing an increase of cell motility and invasiveness in this cancer [κB signaling activation induces also HCC metastasis [α interfering with its production by destabilizing its mRNA [α, and VEGF by modulating their expression [κB and its subunit, RELA, are correlated with IL-1, IL-8, and TNF-α that interacts with IL-7 creating a heterodimer that contains also their receptors. Moreover, it is very interesting that RELA interacts also with BECN1; in fact, it modulates its transcription and autophagy upregulating BECN1 mRNA and protein levels in different cellular systems [κB complex, evidencing that the decrease of their levels can induce NF-κB inactivation able to block HCC development, progression, and metastasis.In detail, five significant molecules are connected with some hub proteins, such as NF-s cancer , (ii) STs cancer , and (iitastasis . In detapression . Finally systems . OverallIn conclusion, our results have shown that doxorubicin and tacrolimus inhibit the growth of both hepatocellular carcinoma cell lines, Huh7 and HepG2, in a dose-dependent manner and their combination induces a more cytotoxic effect. Therefore, we suggest that the mechanisms involved could be a apoptosis induction, contemporaneous decrease of autophagy activation, understood as prosurvival process and immunosurveillance decrement, as well as downregulation of cytokine production. Moreover, it is important to underline that the concentrations used during the cotreatment were lower than IC50 obtained by dose-response assays with each drug treatment. In conclusion, our results indicate that tacrolimus in combination with doxorubicin is expected to be a support drug in HCC treatment. However, these experiments were obtained on cell lines and future studies will regard in vivo experiments to verify if the doxorubicin/tacrolimus combination can have effects in mice models. Until now, there are few studies in this field; in fact, it is resulted in that doxorubicin has improved antitumor activity both in vitro and in vivo with much reduced side effects , whereas


To assess the prevalence of glaucoma in rural Central India.The population-based Central India Eye and Medical Study is a population-based study performed in a rural region of Central India. The study included 4711 subjects (aged 30+ years). A detailed ophthalmic and medical examination was performed. Glaucoma was defined by glaucomatous optic disc morphology, and in a second step, by the criteria of the International Society of Geographical and Epidemiological Ophthalmology (ISGEO).P<0.001), lower body mass index (P = 0.025), lower blood hemoglobin concentration (P = 0.03), higher intraocular pressure (P<0.001), disc hemorrhages (P<0.001), higher prevalence of myopic retinopathy (P<0.001), lower level of education (P = 0.03), longer axial length (P<0.001), thinner retinal nerve fiber layer (P<0.001), higher vertical cup/disc diameter ratio (P<0.001), and narrow anterior chamber angle (P = 0.02). Ratio of open-angle glaucoma to angle-closure glaucoma was 7.7:1  to 0.24% ). Using the ISGEO criteria, glaucoma prevalence was 2.8%  with a less clear association with older age.Optic disc photographs were available for 4570 (97.0%) subjects. Glaucoma was detected in 122 subjects  . Glaucoma prevalence for the age groups of 30–39yrs, 40–49yrs, 50–59yrs, 60–69yrs, 70–79yrs, and 80+ years was 0.54% , 1.03% , 1.40% , 6.62% , 8.71% , and 14.3% , respectively. In multivariable analysis, glaucoma was associated with higher age (Glaucoma prevalence in remote rural Central India is comparable to other regions. Associated factors were older age, lower body mass index, lower blood concentration of hemoglobin, lower level of education, higher intraocular pressure, disc hemorrhage, myopic retinopathy, and longer axial length. The ratio of open-angle glaucoma to angle-closure glaucoma was about 8:1. Knowledge about the prevalence of a disease and the associated factors is instrumental for an improved detection of the disorder and for a better understanding of its pathogenesis. Factors associated with the development of glaucoma or with its further progression have been examined in several population-based studies and in numerous large hospital-based investigations The Medical Ethics Committee of the Medical Faculty Mannheim of the Ruprecht-Karls-University Heidelberg and the Ethics Committee of the Suraj Eye Institute/Nagpur approved the study; all participants gave informed written consent, according to the Declaration of Helsinki.The Central India Eye and Medical Study (CIEMS) is a population-based cross-sectional study performed in Central India between 2006 and 2008. As described in detail previously, the study was performed in 8 remote villages in Kalmeshwar Tehsil, a rural region of Eastern Maharashtra at a distance of about 40 km from Nagpur in the geographical center of India All examinations were carried out at the hospital. Trained social workers filled out a questionnaire for the participants; this questionnaire included questions regarding socioeconomic background and living conditions, tobacco use and alcohol consumption, and any known diagnosis of major systemic diseases. In all subjects, the pulse, arterial blood pressure, and body height and weight were recorded. One-and-a-half hours after a standardized lunch, blood and urine samples were obtained and biochemically analyzed. A detailed ophthalmologic examination included testing of visual acuity by ophthalmologists or optometrists. Uncorrected visual acuity and visual acuity with the subjectś glasses and after refractive correction were measured using modified Early Treatment of Diabetic Retinopathy Study (ETDRS) charts  at a distance of 4 meters. Automated refractometry and subjective refraction were performed on all subjects independent of visual acuity. Perimetric examinations were performed with frequency-doubling perimetry using the screening program C-20-1 . Intraocular pressure was measured by a slit lamp mounted Goldmann applanation tonometer; if the measurements were higher than 21 mmHg, tonometry was repeated. Slit lamp biomicroscopy was carried out by a fellowship-trained ophthalmologist, and any abnormality of the anterior segment was noted. With the subject in the supine position, corneal pachymetry and biometry were carried out via sonography using the Pacscan . Central corneal thickness, anterior chamber depth, lens thickness and axial length were measured on each eye of all subjects. The examinations were performed up to five times each, and the mean values were taken. In subjects with poor visual acuity and poor fixation or in highly myopic subjects with posterior staphyloma, if the measurements varied, they were repeated until reproducible measurements were obtained. Using the slit lamp, photographs of the limbal region were taken to assess the limbal anterior chamber depth at the most peripheral part of the cornea, as described by van Herick Digital photographs of the lens were taken and nuclear sclerosis was graded according to the Age Related Eye Disease Study Digital photographs of the optic disc (20 degrees) and photographs centered on the macula and the optic disc (50 degrees) using the Zeiss FF450 fundus camera were taken. Magnification by optic media was corrected for by a built-in algorithm. Using the planimetric analysis system of the fundus camera, we outlined the margins of the optic disc and optic up on the digitized photographs and measured the area and horizontal diameter and vertical diameter of the optic disc and cup. Glaucoma was defined by a glaucomatous appearance of the optic disc. The optic nerve head was glaucomatous (1) if the inferior-superior-nasal-temporal (ISNT)-rule of the neuroretinal rim shape was not fulfilled in early glaucoma and in eyes with a normally shaped optic disc ; or (2) if an abnormally large cup was present in a small optic disc which normally would not show cupping. In all eyes with glaucoma, the visibility of the retinal nerve fiber layer was locally and/or segmentally reduced. The assessment of the optic disc photographs was carried in a masked manner without knowledge of intraocular pressure or the perimetric results. Each photograph of a glaucomatous optic disc was independently adjudicated by two senior graders (VN and JBJ). In addition to the optic disc photographs, confocal laser scanning tomograms  of the optic disc were taken.The whole glaucoma group was differentiated into subjects with open-angle glaucoma and with primary angle closure glaucoma. Open-angle glaucoma was characterized by an open anterior chamber angle, in addition to a normal depth of the anterior chamber as assessed by slit lamp biomicroscopy. In angle-closure glaucoma, the anterior chamber angle was occluded or occludable. The anterior chamber angle was defined as occludable, if ≥270° of the posterior trabecular meshwork could not be seen upon gonioscopy Fundus photographs were graded for signs of myopic retinopathy which was defined as the presence of at least 1 of the following features: staphyloma, lacquer cracks, Fuchs’ spot, or chorioretinal atrophy at the posterior pole P-values were 2-sided and were considered statistically significant when the values were less than 0.05. In the analysis, we first calculated the mean values in the study population. In a second step, we examined the association between the prevalence of glaucoma and other factors in univariate analysis. In a third step, we performed a multivariable binary regression analysis with the presence of glaucoma as dependent variable and all variables as independent parameter, which were significantly associated with the prevalence of glaucoma in the univariate analysis and which could be regarded to be causally related to glaucoma, instead of being a consequence of it. We then excluded step by step those variables from the list of independent parameters which were no longer significantly associated with the prevalence of glaucoma, starting with the parameters with the highest P-values. In a fourth step of the statistical analysis, we added to the list of independent parameters those variables which may have been changed by glaucoma. In a fifth step, we assessed the prevalence of glaucoma as defined by the criteria of the International Society of Geographic and Epidemiological Ophthalmology ISGEO Only those subjects with assessable optic disc photographs were included into the study. The status of the lens was no inclusion or exclusion criterion. Statistical analysis was performed using a commercially available statistical software package . Continuous data were presented as mean ± standard deviation. Chi-square tests were used to compare proportions. Logistic or linear regression models were used to investigate the associations of the presence of glaucoma with the continuous  or categorical outcomes . All P<0.001), was significantly more myopic , had significantly longer axial length , and had significantly more women . Intraocular pressure did not vary significantly between both groups .Out of the 9422 eyes (4711 subjects), optic disc photographs were available for 8869 (94.1%) eyes of 4570 (97.0%) subjects. The other 141 participants were excluded since assessable photographs of the optic nerve head were not available. The mean age of the 4570 participants (53.8% women) was 48.5±12.9 years , with 23.8% being within the age group of 30 to 39 years, 29.2% in the group of 40 to 49 years, 17.1% in the group of 50 to 59 years, 19.1% in the group of 60 to 69 years, and 11.0% in the age group of 70+ years. The mean refractive error was –0.06±1.70 diopters , and mean axial length was 22.64±0.86 mm . The group of subjects without optic disc assessment as compared with the group of subjects included into this study was significantly older . The prevalence of glaucoma per person for the age groups of 30–39 years, 40–49 years, 50–59 years, 60–69 years, 70–79 years, and 80+ years was 0.54% , 1.03% , 1.40% , 6.62% , 8.71% , and 14.3% , respectively  of 122 subjects  . In univariate analysis, the glaucoma frequency increased significantly with older age (ectively . For theP = 0.03), lower body weight (P<0.001), lower body mass index (P<0.001), higher systolic (P<0.001) and diastolic (P<0.001) blood pressure and prevalence of arterial hypertension (P<0.001), lower level of education (P<0.001), lower blood concentration of hemoglobin (P = 0.03), and with the ocular parameters of higher intraocular pressure (P<0.001), higher ocular perfusion pressure (P = 0.03), longer axial length (P<0.001), more myopic refractive error (P<0.001), higher degree of nuclear cataract (P<0.001), narrower opening of the anterior chamber angle as assessed by gonioscopy (P = 0.001), higher anterior chamber angle pigmentation (P<0.001), higher presence of pseudoexfoliation (P<0.001), larger optic disc (P = 0.001) and optic cup (P<0.001), smaller neuroretinal rim (P = 0.003), higher horizontal and vertical cup/disc diameter ratios (P<0.001), lower retinal nerve fiber layer cross section area as measured by the HRT (P<0.001), lower visibility of the macular wall reflex (P = 0.006), higher prevalence of myopic retinopathy (P<0.001), and higher prevalence of optic disc hemorrhages (P<0.001). The prevalence of glaucoma was not significantly associated with the systemic parameters of gender (P = 0.07), pulse rate (P = 0.10), score of psychic depression (P = 0.98), blood concentration of high-density lipoproteins (P = 0.95), cholesterol (P = 0.16), creatinine (P = 0.98), glycosylated hemoglobin HbA1c (P = 0.92), presence of diabetes mellitus (P = 0.46), erythrocyte sedimentation rate (P = 0.16), drinking alcohol (P = 0.19), and with the ocular parameters of anterior corneal curvature (P = 0.79), central corneal thickness (P = 0.07), anterior chamber depth (P = 0.91), lens thickness (P = 0.28), prevalence of retinal vein occlusions (P = 0.50), presence of central retinal artery occlusion (P = 1.00), presence of diabetic retinopathy (P = 1.00), presence of early age-related macular degeneration (P = 0.34), presence of myelinated retinal nerve fibers (P = 0.52).The prevalence of glaucoma was significantly additionally associated with lower body height , ocular perfusion pressure and degree of nuclear cataract (P = 0.66), presence of pseudoexfoliation (P = 0.95), systolic blood pressure (P = 0.66), diastolic blood pressure (P = 0.61), and body height (P = 0.13), prevalence of glaucoma remained to be significantly associated with older age (P<0.001), lower body mass index (P = 0.01), lower blood concentration of hemoglobin (P = 0.001), higher intraocular pressure (P<0.001), higher mean arterial blood pressure (P = 0.002), presence of a disc hemorrhage (P<0.001), higher prevalence of myopic retinopathy (P<0.001), longer axial length (P = 0.004), larger optic disc size (P<0.001), lower level of education (P = 0.03), and more narrow anterior chamber angle (P = 0.01).In a first step of a multivariable analysis, a binary regression analysis was performed with the presence of glaucoma as dependent variable and all the variables as independent parameters, which were significantly associated with the prevalence of glaucoma in the univariate analysis and which could be regarded to be causally related to glaucoma . The list of independent parameters included age, body height, body weight, body mass index, systolic and diastolic blood pressure, level of education, blood concentration of hemoglobin, intraocular pressure, ocular perfusion pressure, axial length, refractive error, degree of nuclear cataract, opening of the anterior chamber angle, anterior chamber angle pigmentation, presence of pseudoexfoliation, optic disc area, prevalence of myopic retinopathy and prevalence of optic disc hemorrhages. We then excluded step by step those variables from the list of independent parameters which were no longer significantly associated with the prevalence of glaucoma, starting with the parameters with the highest P-value). After a step-wise removal of the visibility of the macular wall reflex (P = 0.99), horizontal cup/disc ratio (P = 0.25), optic disc area (P = 0.90), optic cup area (P = 0.14) and finally neuroretinal area (P = 0.08), one arrived at a model in which glaucoma was significantly associated with older age (P<0.001), lower body mass index (P = 0.025), lower blood concentration of hemoglobin (P = 0.03), higher intraocular pressure (P<0.001), higher mean blood pressure (P = 0.009), presence of a disc hemorrhage (P<0.001), higher prevalence of myopic retinopathy (P<0.001), lower level of education (P = 0.03), longer axial length (P<0.001), smaller retinal nerve fiber layer cross section area (P<0.001), higher vertical cup/disc diameter ratio (P<0.001), and narrowness of the anterior chamber angle (P = 0.02) (P = 0.69) associated with the glaucoma prevalence.We then added to the list of independent parameters those variables which may have been changed by glaucoma. These were the variables of neuroretinal rim area, optic cup area, horizontal and vertical cup/disc diameter ratios, retinal nerve fiber layer cross section area as measured by the HRT, and visibility of the macular wall reflex. We then removed the parameters which showed the least likely association with glaucoma  . If we aP = 0.005), higher intraocular pressure (P<0.001), higher prevalence of disc hemorrhages (P<0.001), more hyperopic refractive error (P = 0.003), shallower anterior chamber (P = 0.02), shorter axial length (P = 0.01) and higher vertical cup/disc ratio (P<0.001), while it was not significantly associated with the level of education (P = 0.93), presence of myopic retinopathy (P = 0.99), mean blood pressure (P = 0.23), blood concentration of hemoglobin (P = 0.20), and body mass index (P = 0.21) ; 172 eyes) and subjects with primary angle-closure glaucoma (n = 14 (11.5%); 21 eyes). Prevalence of open-angle glaucoma was 1.93% , and prevalence of angle-closure glaucoma was 0.24% . In multivariable analysis, the prevalence of open-angle glaucoma was significantly associated with the similar parameters as was the whole glaucoma group associated with . In cont = 0.21) .In the whole study population, cataract surgery had been performed in 273 eyes. Within this group of eyes after cataract surgery, 37 (13.6%) eyes were classified to have glaucoma, with 34 (92%) subject having open-angle glaucoma and 3 (8%) eyes having angle-closure glaucoma. This ratio of 11 to 1 was higher than the ratio in the whole study population (108/14 or 7 to 1).According to the World Health Organization criteria, low vision and blindness were defined as visual acuity in the better seeing eye as <20/60 to 20/400, and as <20/400, respectively. In the 108 subjects with open-angle glaucoma, 34 (31.5%) subjects had low vision due to glaucoma, and one (0.9%) of the subjects with open-angle glaucoma was blind. Unilateral visual impairment was present in 65 (37.8%) of the 172 eyes with open-angle glaucoma, and glaucoma related blindness occurred in 18 (10.5%) eyes with open-angle glaucoma. Out of 14 subjects with primary angle-closure glaucoma, 3 (21%) subjects had low vision and none of the subjects was blind due to glaucoma. Unilateral visual impairment was present in 7 (33%) of the 21 eyes with angle-closure glaucoma, and glaucoma related blindness occurred in 4 (19%) eyes with angle-closure glaucoma.The mean vertical cup/disc diameter ratio was 0.55±0.12 (median: 0.56) . The 97.P = 0.25) associated with higher age  of 130 subjects  . The number of subjects in each diagnostic category is listed in gher age . In viewIn our population-based in rural Central India, glaucoma was detected in 2,67% of the subjects, in whom open angle glaucoma (OAG) and primary angle closure glaucoma (PACG) accounted for 1.93% and 0.24%. In multivariable analysis, glaucoma was associated with older age, lower body mass index, lower blood concentration of hemoglobin, higher intraocular pressure, higher mean blood pressure, presence of a disc hemorrhage, higher prevalence of myopic retinopathy, lower level of education, longer axial length, smaller retinal nerve fiber layer cross section area, higher vertical cup/disc diameter ratio, and narrowness of the anterior chamber angle .For India, the frequency of glaucoma has been reported by the Vellore Eye Survey, Andhra Pradesh Eye Disease Study, Aravind Comprehensive Eye Survey, Chennai Glaucoma Study, and West Bengal Glaucoma Study Our study showed a ratio of open-angle glaucoma to angle-closure glaucoma of 108/14 or 7.7:1. This ratio is similar to the ratio reported in some other Asian studies such as the Japanese Tajimi study (3.9% to 0.6% or 6.5:1) P<0.001), lower body mass index (P = 0.025), lower blood concentration of hemoglobin (P = 0.03), higher intraocular pressure (P<0.001), higher mean blood pressure (P = 0.009), presence of a disc hemorrhage (P<0.001), higher prevalence of myopic retinopathy (P<0.001), lower level of education (P = 0.03), longer axial length (P<0.001), smaller retinal nerve fiber layer cross section area (P<0.001), higher vertical cup/disc diameter ratio (P<0.001), and narrowness of the anterior chamber angle (P = 0.02)  . The ageP = 0.07) nor in multivariable analysis (P = 0.69). Since most of the glaucoma patients in our study population were not aware of their disease, a confounding factor may be unlikely. It suggests that central corneal thickness may not be related to glaucoma susceptibility, what agrees with, or is contradictory to some previous studies Central corneal thickness was associated with the glaucoma prevalence neither in univariate analysis (Presence of myopic retinopathy and in addition, long axial length were factors associated with the glaucoma prevalence. These findings confirm results from previous studies on other populations in which high myopia was a highly significant risk factor for glaucomatous optic neuropathy In our study, the glaucoma definition based on the subjective assessment as compared to the definition based on the ISGEO criteria of the optic nerve head appearance resulted in apparently more reasonable results. In many recent population-based investigations on the prevalence of glaucoma such as the Liwan Eye Study the ISGEO definition of glaucomatous optic neuropathy has usually been applied, based on a consensus published by Foster and colleagues Potential limitations of our study should be discussed. First, a major concern in any prevalence study is nonparticipation. The Central India Eye and Medical Study had a reasonable response rate of 80.1%, however, differences between participants and non-participants can lead to a selection artifact. Second, our study included only those who resided in a purely rural region, a region that can be considered to be markedly rural based on responses to the questionnaire regarding socioeconomic background and lifestyle. The study did not include subjects from an urban region, so we can provide no information on any differences between rural and urban regions with respect to the examined parameters. Third, our study as cross-section investigation does not allow firm statements on a longitudinal association, i.e. temporal causation cannot be established with the current cross-sectional design. Fourth, it could not be determined whether disk hemorrhages were associated with glaucoma or were a result of posterior vitreous detachment or other vascular pathology In conclusion, glaucoma prevalence in remote rural Central India was comparable to other regions. It increased with older age, lower body mass index, lower blood concentration of hemoglobin, higher intraocular pressure, presence of an optic disc hemorrhage, higher prevalence of myopic retinopathy, lower level of education, longer axial length, smaller retinal nerve fiber layer cross section area, and higher vertical cup/disc diameter ratio. The ratio of open-angle glaucoma to angle-closure glaucoma was about 8:1.