Several reports have revealed potent We analyzed the effects of neonatal exposure to BPA on the reproductive axis of female Sprague-Dawley rats.in vivo and in vitro, as well as gonadotropin-releasing hormone (GnRH) pulsatility at PND13 and in adults. We also analyzed two GnRH-activated signaling pathways in the adults: inositol-triphosphate (IP3), and extracellular signal-regulated kinase1/2 (ERK1/2).Female rats were injected subcutaneusly, daily, from postnatal day 1 (PND1) to PND10 with BPA [500 μg/50 μL (high) or 50 μg/50 μL (low)] in castor oil or with castor oil vehicle alone. We studied body weight and age at vaginal opening, estrous cycles, and pituitary hormone release in vivo was decreased and GnRH pulsatility remained disrupted. In vitro, pituitary cells from animals treated with BPA showed lower basal LH and dose-dependently affected GnRH-induced IP3 formation; the high dose also impaired GnRH-induced LH secretion. Both doses altered ERK1/2 activation.Exposure to BPA altered pituitary function in infantile rats, lowering basal and GnRH-induced luteinizing hormone (LH) and increasing GnRH pulsatility. BPA dose-dependently accelerated puberty onset and altered estrous cyclicity, with the high dose causing permanent estrus. In adults treated neonatally with BPA, GnRH-induced LH secretion in vivo BPA permanently affects GnRH pulsatility and pituitary GnRH signaling.Neonatal exposure to BPA altered reproductive parameters and hypothalamic–pituitary function in female rats. To our knowledge, these results demonstrate for the first time that neonatal  In contg/kg/day , Wistar g/kg/day , and Fisg/kg/day . These pg/kg/day .In addition to its estrogenicity, BPA can antagonize the effects of estrogens, androgens, or thyroid hormones; act through non-genomic pathways; and influence enzyme activity or receptor expression .Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide critical for normal mammalian reproductive function. GnRH secretion is pulsatile and acts on gonadotropes to stimulate synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) . Gonadotq/11-coupled receptor), phospholipase C is activated, leading to diacylglycerol and inositol 1,4,5-triphosphate (IP3) formation  and protein kinase C (PKC) is activated, resulting in multiple cellular responses, such as activation of the mitogen-activated protein kinase (MAPK) family (After binding of GnRH to its receptor (a Gormation . Subsequ) family .in vivo and in vitro pituitary response to GnRH, GnRH pulsatility, and GnRH-activated signaling pathways.In this study we explored the effects of neonatal exposure to high and low doses of BPA on reproductive parameters in female Sprague-Dawley rats, a strain reported to have low sensitivity to estrogenic compounds . We analStudies were performed according to protocols for animal use  and apprSprague-Dawley rats (200–250 g) from the IByME colony, established from Charles River stock in 1985, were maintained under a controlled 12-hr light/dark cycle and temperature conditions. They were housed in steel cages with wood shavings as bedding material and given free access to commercial laboratory chow  and tap water in glass bottles. We did not test the phytoestrogen concentration in the food, but we assumed that all animals were exposed to the same levels, since food intake was equivalent between groups.Pregnant females were housed individually; on the day of birth [postnatal day (PND) 1], litters were reduced to eight pups. Female neonates from each litter were assigned to the different experimental groups and left with the dam. On PND1–PND10, each pup received a daily subcutaneous injection of BPA  in castor oil  or castor oil vehicle (control). BPA500 has been used previously in this and other strains of rats , 2007; bOn PND13, an age in which the pituitary is highly sensitive to stimulatory and inhibitory inputs , animalsOn PND13, we injected 9–12 animals from each group intraperitoneally with 100 ng GnRH  or saline solution. After 15 min (LH) or 50 min (FSH), trunk blood was collected; serum LH and FSH were then determined by RIA.ex vivo as described by We performed pulsatility studies http://mljohnson.pharm.virginia.edu/home.html). We used a 2 × 2 cluster configuration and a t statistic of 2 for the upstroke and downstroke to maintain false-positive and false-negative error rates < 10%, as suggested by We identified GnRH pulses and defined their parameters using Cluster8 computer algorithm analysis developed by −9 M and 1.10−7 M (1 hr). Media were stored (−20°C) for gonadotropin analysis by RIA. The experiment was performed in quadruplicate (n = 7 per treatment group).Using high-dose animals and controls on PDN13, we obtained anterior pituitary cells as described by After weaning on PND21, 11–15 pups from each treatment group  were weighed daily and the age and weight at vaginal opening (VO) were recorded. Animals were multiple-housed (four per cage), keeping members of each litter together. We determined estrous cycles in five animals from each group from PND60 to PND120 by examining vaginal smears under a light microscope. After PND120, serum hormones, pituitary response to GnRH, and GnRH pulsatility were determined in animals in estrus.For this experiment we used seven adult animals from each treatment group. Under ketamine/xylazine anesthesia (60/10 mg/kg body weight), one blood sample was collected from the jugular vein (0 min) and GnRH (100 ng) was administered into the same vein. Further samples were taken at 15 and 50 min. Serum LH was determined by RIA.n = 7).Experiments and analysis were performed as described above (1), biphosphate (IP2), and IP3 were measured as described by 3H(N)]-myo-inositol , washed with SFM, and incubated for 15 min in the presence of 20 mM lithium chloride. Stimuli were added , and cells were incubated for 30 min. After incubation, media were collected to measure LH, and cells were scraped in 0.5% perchloric acid; lysates were then transferred to microfuge tubes, neutralized with 0.72 M potassium hydroxide and 0.6 M potassium bicarbonate, and centrifuged for 10 min at 3,000 rpm. Supernatants were chromatographed in ionic exchange columns  to elute free inositol, IP1, IP2, and IP3. Aliquots of the eluates were mixed with OptiPhase “Hisafe” 3 (PerkinElmer) and counted in a liquid scintillation counter; results are expressed as IP3/inositol (n = 6 per treatment group).Levels of inositol monophosphate . Media were then removed and sample buffer (80 μL) added to the wells; plates were stored at −70°C. Cell lysates (20 μL) were run on 12% SDS-PAGE gels (Bio-Rad) and electro transferred to nitro-cellulose membranes, which were then blocked with 5% BSA–0.1% Tween-phosphate buffered saline (TPBS) for 2 hr at room temperature (RT). Membranes were incubated with a mouse phospho-specific ERK1/2 antibody  for 2 hr at RT. Membranes were stripped, blocked with 3% fat-free milk–TPBS buffer (1 hr at RT), and probed for 1 hr at RT with a rabbit polyclonal antibody that detects total ERK1/2 . Signals were detected with horseradish peroxidase-conjugated anti-mouse antibody  or anti-rabbit antibody  and visualized by chemiluminescence. Immunoblots were quantified using Scion Image Software ; results are expressed as pERK1/2/total ERK1/2 (n = 4 per treatment group).We determined ERKanalysis . PituitaWe determined hormones by RIA using kits obtained from the National Hormone and Peptide Program . Results are expressed in terms of reference preparation 3 rat LH, FSH, and PRL standards. Assay sensitivities were 0.015 ng/mL for LH, 0.1175 ng/mL for FSH, and 0.04 ng/mL for PRL. Intraassay and interassay coefficients of variation, respectively, were as follows: LH, 7.2% and 11.4%; FSH, 8.0% and 13.2%; and PRL, 8.1% and 11.4%. We performed the GnRH RIA as described by 3/inositol, Western blots, and LH and FSH released to culture media by repeated measures two-way ANOVA . Data were transformed when the test for homogeneity of variances so required. Results are expressed as mean ± SE, and p < 0.05 is considered significant.We analyzed hormonal levels in PND13 and adult animals, pulsatility and puberty parameters, and estrous cycles by one-way or two-way analysis of variance (ANOVA) using Statistica, version 5, software . We analyzed GnRH-induced LH in adults, IPOn PND13, BPA500-treated animals had significantly lower basal serum LH than did controls . GnRH significantly increased serum LH in all groups, but exposure to BPA dose dependently lowered GnRH-induced LH release, reaching statistical significance at the higher dose . However−9 M and 1.10−7 M) stimulation, similar to the in vivo results. For 1.10−7 M GnRH, LH concentrations were as follows: for control, 17.37 ± 1.66 ng/50,000 cells for basal and 46.75 ± 7.81 ng/50,000 cells for GnRH; for BPA500, 7.67 ± 0.87 ng/50,000 cells for basal and 24.43 ± 4.08 ng/50,000 cells for GnRH . Again, percent increases were similar between groups for LH; after 1.10−7 M GnRH, percent increases were 277.4 ± 56.3% for control and 334.3 ± 68.0% for BPA500 (not significant). We observed no differences in basal or GnRH-induced FSH levels between groups (data not shown).Next, we evaluated the response to GnRH in pituitary cells from control and high-dose animals cultured in vitro, a situation in which cells were deprived from the regulatory inputs from the gonads and the brain. Pituitary cells from the BPA500 group released significantly less LH to the media than controls, basally and after GnRH .Because we found differences in GnRH pulsatility with these two BPA doses, we carried out another experiment using hypothalami from animals injected as before but with a 10-fold lower dose of BPA (5 μg) (BPA5). Interestingly, we found that BPA5 animals also exhibited significantly higher pulse frequency than controls: 0.46 ± 0.04 peaks/hour for controls, and 0.83 ± 0.10 peaks/hr for BPA5 (p < 0.05). We found no significant differences in body weight among the three groups .In adulthood, only animals from the the BPA500 group showed irregular estrous cycles, with high prevalence of estrus after PND90, whereas proestrus and diestrus were markedly reduced. The percentage of days (± SE) in each stage of the cycle for the three groups are as follows: control: diestrus = 49.5 ± 0.5%, proestrus = 25.0 ± 0.8%, estrus = 25.5 ± 1.2%; BPA50: diestrus = 50.8 ± 5.1%, proestrus = 22.7 ± 3.2, estrus = 25.8 ± 2.7; BPA500: diestrus = 0.8 ± 0.8%, proestrus = 1.6 ± 0.9%, estrus = 97.7 ± 1.5%;  were 3.15 ± 0.65 ng/mL for control, 4.66 ± 0.41 ng/mL for BPA500, and 3.47 ± 0.47 ng/mL for BPA50; FSH concentrations were 5.67 ± 0.90 ng/mL for control, 6.18 ± 0.55 ng/mL for BPA500, and 6.98 ± 0.44 ng/mL for BPA50. As shown in in vivo GnRH injection, serum LH increased in all groups; levels were lower in both BPA-treated groups, but this attained statistical significance only in high-dose animals. After 50 min of stimulation, LH was still increased, without differences among groups. Interestingly, in both the control and BPA50 groups at 50 min, LH levels were significantly lower than those at 15 min, whereas in the BPA500 group, LH levels did not vary between these time points.We also studied pituitary function in adulthood, both n = 7; control vs. BPA50 and BPA500, p < 0.05].Regarding GnRH pulsatility, adult animals neonatally exposed to either dose of BPA exhibited higher pulse frequency than controls, as demonstrated in PND13 females: control, 0.60 ± 0.05 peaks/hr; BPA50, 0.81 ± 0.03 peaks/hr; BPA500, 0.83 ± 0.04 peaks/hr .In addition, we determined GnRH responsiveness controls . Cells fcontrols . In all 1/2 pathway in these experimental groups (1/2 phosphorylation was rapid (apparent after 5 min of GnRH stimulation) and sustained (still significantly activated after 30 min), as classically reported ; BPA50, 5.42 ± 0.66 ng/mL (n = 7); BPA500, 11.34 ± 1.31 ng/mL (n = 8); BPA500 vs. control, p < 0.05].We observed no significant differences in serum PRL between groups on PND13 (data not shown). Nevertheless, serum PRL was significantly higher in BPA500 adult animals than in controls, and levels in BPA50 animals were similar to those in controls [control, 4.34 ± 0.67 ng/mL  increased GnRH pulse frequency, with an increase in the number of peaks per hour. Similar effects on GnRH pulsatility have been demonstrated by estradiol and DDT administration . When tested stration . The decstration , althougNeonatal treatment with high and low doses of BPA also affected puberty onset, dose-dependently advancing VO, without affecting body weight. A similar advance in puberty onset has also been described after a single injection of estradiol on PND10  or afterPuberty changes occur as a consequence of the activation of the hypothalamic–pituitary–gonadal axis . As discIn adults the higher dose significantly disrupted estrous cycles with a high prevalence of estrus. Although we found no effect on estrous cycles with the lower dose (BPA50), we cannot exclude the possibility of loss of cyclicity later in life. in vivo GnRH-induced LH release in adult females, with BPA500 reducing the response to the decapeptide; to our knowledge, this is the first report of such an effect. In addition, we also observed accelerated GnRH pulse frequency in adult females neonatally exposed to high and low BPA, demonstrating that the effect found in infantile animals persists throughout life. Moreover, adult pituitary cells from animals neonatally exposed to BPA showed decreased basal LH release, but only the pituitaries from high-dose animals released less LH after GnRH stimulation. When analyzing signal transduction pathways elicited by GnRH, we found that in pituitary cells from BPA-treated animals, GnRH-stimulated IP3 formation was impaired, showing dose dependency for this effect and tightly correlating with LH secretion. This is in agreement with the concept that the IP3–calcium pathway is involved in gonadotropin release. In contrast, we found that the pattern of ERK1/2 activation after GnRH stimulation was altered in cultures from both BPA treatment groups. Controls showed the classical rapid and sustained activation described in gonadotropes (1/2 pathway links signals from the cell surface to the cytoplasm and nucleus; this results in transcriptional and mitogenic events. In the case of GnRH-induced ERK activation, this pathway is involved in LHβ (1/2 phophorylation than the other groups; this is in line with the results obtained with IP3, because activation of ERK1/2 is downstream to PKC activation (In the present study, neonatal treatment with BPA did not modify basal serum gonadotropins but did have an impact on the dotropes , whereasd in LHβ  and GnRHd in LHβ  and probd in LHβ , among otivation . These rWe found no differences in serum PRL titers in infantile animals after BPA treatment. In contrast, BPA500 animals showed hyperprolactinemia. These results agree with a previous study showing hyperprolactinemia at PND20 and PND30 but not PND15 . Other sTaken together, these results demonstrate that exposure to high and low doses of BPA, an environmental endocrine disruptor, in a period of time critical for development alters reproductive parameters in infantile and adult animals, causing precocious hypothalamic–pituitary maturation and precocious puberty, altering GnRH pulsatility in infantiles and adults, and severely affecting GnRH signaling in the adult pituitary. More studies are needed, including those with additional doses of BPA, to further understand the mechanisms underlying these effects.


P = 0.173 for the 4 h and 12%, P = 0.051 for the 24 h time interval). In H460 cells, paclitaxel increased 2-fold the accumulation of dFdCTP, the active metabolite of gemcitabine, in contrast to H322 cells. Paclitaxel did not affect deoxycytidine kinase levels, but ribonucleotide levels increased possibly explaining the increase in dFdCTP. Paclitaxel did not affect gemcitabine incorporation into DNA, but seemed to increase incorporation into RNA. Gemcitabine almost completely inhibited DNA synthesis in both cell lines (70–89%), while paclitaxel had a minor effect and did not increase that of gemcitabine. In conclusion, various gemcitabine–paclitaxel combinations did not show sequence dependent cytotoxic effects; all combinations were not more than additive. However, since paclitaxel increased dFdCTP accumulation, gemcitabine incorporation into RNA and the apoptotic index, the administration of paclitaxel prior to gemcitabine might be favourable as compared to reversed sequences. © 2000 Cancer Research CampaignGemcitabine and paclitaxel are active agents in the treatment of non-small-cell lung cancer (NSCLC). To optimize treatment drug combinations, simultaneously and 4 and 24 h intervals, were studied using DNA flow cytometry and multiple drug effect analysis in the NSCLC cell lines H460, H322 and Lewis Lung. All combinations resulted in comparable cytotoxicity, varying from additivity to antagonism (combination index: 1.0–2.6). Gemcitabine caused a S (48%) and G1 (64%) arrest at IC-50 and 10 × IC-50 concentrations, respectively. Paclitaxel induced G2/M arrest (70%) which was maximal within 24 h at 10 × IC-50. Simultaneous treatment increased S-phase arrest, while at the 24 h interval after 72 h the first drug seemed to dominate the effect. Apoptosis was more pronounced when paclitaxel preceded gemcitabine  as compared to the reverse sequence (8%,


To determine whether activity of carbohydrate metabolism enzymes  are altered in the glaucomatous trabecular meshwork (TM) compared to controls.Tissue specimens were obtained from trabeculectomy  and from cadaver eyes . Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm.Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS–PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls.Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity. Open angle glaucoma is an irreversible late onset and progressively blinding disease. The anatomy of anterior chamber angle, the site of aqueous egress, between iris and cornea, has been used to differentiate open angle glaucomas from angle closure glaucomas. Primary open angle glaucoma (POAG) is established as a way of exclusion when no inciting condition can be ascribed to development of open angle glaucoma. Glaucomas are termed secondary when they occur in association with prior injury or concomitant illness. Elevated intraocular pressure (IOP) is a risk factor for glaucoma.Glaucomas have been ascribed to an imbalance in aqueous humor production and outflow. Increased resistance at the level of TM decreases the outflow and leads to elevated IOP . In POAGGlaucomatous tissue specimens were obtained from trabeculectomy  and from cadaver eyes . Cadaver eye derived TM tissue (n=15) from non-glaucomatous donors of both genders (age range 59–67) were used as normal controls. Protein extracts from TM tissue were prepared in a non-denaturing buffer  containing 0.1% Genapol C-100 . The research was conducted with local IRB committee approval, following the tenets of the declaration of Helsinki.Aldolase converts fructose-1,6-bisphosphate into dihydroxy acetone phosphate and glyceraldehyde-3-phosphate. Aldolase enzymatic activity assay was measured using the Bayer modification of the hydrazine assay; 3-phosphoglycerate reacts with hydrazine to form hydrazone. Hydrazone absorbs at 240 nm and is quantified spectrophotometrically .Pyruvate kinase catalyzes the conversion of phosphoenol pyruvate plus ADP into pyruvate plus ATP. The reaction kinetics were quantified by coupling with lactate dehydrogenase NADH + pyruvate resulting form lactate + nicotinamide adenine dinucleotide (NAD). Enzyme activity can be measured at 340 nm absorbance by spectrophotometry ,7.Isocitrate dehydrogenase catalyzes the conversion of isocitrate into α-ketoglutarate and carbon dioxide. Simultaneously NADP is reduced into NADPH. Characteristic NADPH absorbance quantifies the enzyme activity at 340 nm ,8. The aMalate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NAD as a coenzyme . Malate t-test.For all activity assays statistical analysis was performed within each group (each group comprised of 10 different tissue samples) and compared to 0.0 using the two tailed one-sample Protein was quantified using the biochinchonic acid (BCA) protein assay . Western blot analysis was performed with 5 µg of protein on 4%–20% Tris-Glycine gels . After fractionation, proteins were electro-blotted onto a polyvinylidene fluoride (PVDF) membrane  using standard procedures and probed with commercially available HNE protein adduct antibody generated in mouse , GAPDH antibody ; aldolase antibody , pyruvate kinase antibody  and polyclonal antibody to isocitrate dehydrogenase . All secondary antibodies were diluted at 1:1,000.To identify the proteins and perform quantification, 80 µg of sample tissue were loaded on to 4%–20% SDS–PAGE gels. Protein bands thus separated were excised, destained with 50% acetonitrile /water, and suspended in 0.5 M triethylammoniumbicarbonate  pH 8.5 and reduced with 10 mM Tris-(2-Carboxyethyl) phosphine . The proteins were subsequently alkylated in the dark using 55 mM solution of iodoacetamide  and in-gel digested with sequencing grade modified trypsin  overnight at 37 °C. The peptides were extracted twice with 50 µl 0.1% triflouroracetic acid/60% acetonitrile and finally with 30 µl of acetonitrile and dried in a SpeedVac. The extracted peptides were incubated with the iTRAQ Rg-8-Plex Assay kit reagents . Separate peptides were isolated from different bands and incubated with reagents 113, 114, 115, and 116  for peptides derived from control TM protein bands, and with reagents 117, 118, 119, and 121 for peptides derived from glaucomatous TM protein bands in 0.5 M TEAB containing 60% v/v isopropanol. The incubation mixtures were dried in a SpeedVac, mixed together and loaded onto a slurry of 500 µl of cation exchange buffer in 12 mM ammonium formate in 25% acetonitrile at pH 2.5–3.0, and separated chromatographically.2D-LC is performed to achieve better separation of peptides before mass spectrometry. Peptides recovered after in-gel digestion were labeled with iTRAQ reagents and then subjected to Strong Cation Exchange (SCX) separation.. SCX peptide separations were performed on a passivated Waters 600E HPLC system, using a 4.6×250 mm polysulfethyl aspartamide column  at a flow rate of 1 ml/min. Buffer A contained 10 mM ammonium formate, pH 2.7, in 20% Acetonitrile/80% water. Buffer B contained 666 mM ammonium formate, pH 2.7, in 20% acetonitrile/80% water. The gradient was Buffer A at 100% (0–22 min following sample injection), 0% to 40% Buffer B (16–48 min), 40% to 100% Buffer B (48–49 min), then isocratic 100% Buffer B (49–56 min), then at 56 min switched back to 100% Buffer A to re-equilibrate for the next injection. The first 26 ml of eluant  was combined into one fraction, then 14 additional 2-ml fractions were collected. SCX fractions were dried down completely to reduce volume and to remove the volatile ammonium formate salts, then resuspended in 9 µl of 2% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid before second dimension separation on reverse phase C18 nanoflow-LC.For the second dimension separation by reverse phase nanoflow LC, each SCX fraction was autoinjected onto a Chromolith CapRod column  using a 5 µl injector loop on a Tempo LC MALDI Spotting system (ABI-MDS/Sciex). Buffer C consisted of 2% acetonitrile, 0.1% trifluoroacetic acid, and Buffer D contained 98% acetonitrile, 0.1% trifluoroacetic acid. The elution gradient was 95% C/5% D , 5% D to 38% D (8.1–40 min), 38% D to 80% D (41–44 min), 80% D to 5% D . Flow rate was 2.5 µl/min during the gradient, and an equal flow of MALDI matrix solution was added post-column , 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile). The combined eluant was automatically spotted onto a stainless steel MALDI target plate every 6 s (0.6 µl per spot), for a total of 370 spots per original SCX fraction.−6 Torr. After the MS and MS/MS spectra from all 15 plates in a sample set were acquired, protein identification and quantitation was performed using the Paragon algorithm as implemented in ProteinPilot 3.0 software  and Matrix Sciences Mascot algorithm version 2.1. ProteinPilot software was used for searches with the following search parameters: Cys Alkylation – Iodoacetamide; ID Focus – Biologic Modifications; Search Effort – Thorough. The Jan 1st 2010 Human NCBI database Sequences containing 512,785 Protein Sequences, plus 156 common laboratory contaminants. For estimation of “False Discovery Rate” (FDR), simultaneous search was performed on a concatenated Decoy database which is the exact reverse of each protein sequence in the database plus 156 common human and laboratory contaminants. Protein identifications at 95% confidence level were retained. The preset “Thorough” (iTRAQ or Identification) search settings were used where identifications must have a ProteinPilot Unused Score >1.3 . In addition, the only protein IDs accepted had a FDR estimation of less than 5%, as calculated from the slope of the accumulated Decoy database hits by the PSPEP  program [The sample spots mentioned above were dryed and thirteen calibrant spots (ABI 4700 Mix) were added to each plate manually. MALDI target plates (15 per experiment) were analyzed in a data-dependent manner on an ABI 4800 MALDI TOF-TOF. As each plate is entered into the instrument, a plate calibration/ MS Default calibration update is performed, and then the MS/MS default calibration is updated. MS spectra were taken from 5,500 MALDI Spots, averaging 500 laser shots per spot at Laser Power 3100. A total of 3,249 MS/MS spectra were taken with up to 2,500 laser shots per spectrum at Laser Power 3600, with CID gas Air at 1.2 to 1.3×10Oxidative stress has been implicated to cause damage to nearly all tissues including trabecular meshwork. Here, we describe that oxidation products may reduce the enzymatic activities of energy metabolism and may become part of the damage process. Thus initial oxidative damage and generated products may have a magnified dampening effect of damage.GDS359) have been performed by several groups using glaucomatous and normal trabecular meshwork tissue. These microarray studies did not show any discernable difference in the expression level of aldolase and pyruvate kinase at the mRNA level. Our quantitative proteomic analysis also showed decreased levels of aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase which is consistent with activity measurement  samples were collected and assayed but only those were included for which details showed definite evidence of ocular health and absence of disease. The trabecular meshwork aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively . Decreassurement . We did Lipid oxidation products have been implicated to cause damage to the aqueous drainage system . Our SDSPreviously, human growth hormone and its interplay with carbohydrate metabolism have been implicated in open angle glaucoma . InvestiIn summary, together with HNE modification of isocitrate dehydrogenase, our results show that energy metabolism enzymes may potentially have reduced activity due to modification or degradation by cellular enzyme cascades in POAG.


Colony formation test and the rate of radiosensitization effect (E/O) were employed to observed the radiosensitization by HSV-TK/GCV system.HSV-TK/GCV suicide gene therapy system and in vitro and in vivo , moreover, the combined administration of gene therapy and radiotherapy had stronger therapeutic effects in vitro and in vivo  (P < 0.01). The inhibition rate by radiotherapy alone was 42.4% in vitro and 35.8% in vivo. The sensitivity of combined therapy to radiotherapy increased more than that of therapy alone, the ability of colony formation decreased (P < 0.01). The rate of radiosensitivity effect (E/O) was 3.17(> 1.4), indicating HSV-TK/GCV system could exert a sensitizing effect on 60Co radiotherapy of the transplanted human cervical cancer cell in nude mice.HSV-TK/GCV suicide gene therapy system had strong therapeutic effects on Hela cells HSV-TK/GCV system had radiosensitization. Gene therapy combined with radiotherapy may be a good supplementary method for cervix cancer synthetic treatment. In recent years, molecular mechanism of tumor development and cell growth regulating mechanism are further studied, gene treatment of tumor has made rapid progress. More attention is paid to HSV-TK/GCV drug sensitive gene treatment system. But its clinical effect is not satisfactory, one of the reasons is that effective dose of GCV (100 mg/kg/1 d) may result in serious toxic side effect. Main complications are damage to renal function (40%~60%) and bone marrow inhibition (20%~41%) . All animal experiments were carried out in compliance with the national laws related to the conduct of animal experimentation.60Co γ ray irradiator , absorptiondose rate was set at 168.5 cGy/min.3cells/well), and cultured for 24 h, when cells grew on wall, they were treated as follows: ① gene treatment group: 10 μg/ml GCV solution was added to each well; ② 60Co radiotherapy group: each well was irradiated by 60Co γ ray, absorption dose was 400 cGy ③ combined treatment group: 10 μg/ml GCV was added to each well, and each well was radiated by 60Co γ ray, absorption dose was 400 cGy ④ control group: no treatment. Each experiment was performed in triplicate. Cells were cultured at 37°C in 5% CO2 incubator for 4 d, then 10 μl (5 mg/ml) MTT solution was added to each well of each group, then cultured for 4 h, finally 150 μl DMSO was added to each well, absorbance value A at 490 nm wavelength was determined, the well without cell was blank, Hela/TK without any treatment was the control, growth inhibition rate of cells was calculated with following formula. Inhibition rate (%) was /absorbance value A of control group × 100%Hela/TK cells were inoculated in 96-well cell culture plate (103 cells/well), and cultured for 24 h, when cells grew on wall, they were treated as follows: ① gene treatment group: 10 μg/ml GCV solution was added to each well; ② 60Co radiotherapy group: each well was irradiated by 60Co γ ray, absorption dose was 400 cGy ③ Combned treatment group: 10 μg/ml GCV was added to each well, and each well was radiated by 60Co γ ray, absorption dose was 400 cGy ④ Control group: no treatment. Each experiment was performed in triplicate. Cells were cultured at 37°C in 5% CO2 incubator for 2 d. Then the cells of control group and experimental group were collected and rinsed in 0.1 PBS (pH 7.2 ~ 7.4) for three times, resuspended and fixed in 70% ethanol at 4 overnight. Cells were centrifuged, resuspended in 0.1 g/L RNase A at 37°C for 30 min and in 0.05 g/L propidium iodide at 4°C for 30 min. Cell cycle was analyzed by flow cytometer .Hela/TK cells were inoculated in 6-well cell culture plate ; ③ simple radiation group, with certain dose of 60Co radiation, absorption dose was 400 cGy; ④ combined treatment group, first 1 μg/ml GCV  was added and then radiated by 60Co, absorption dose was 400 cGy, which were cultured at 37°C in 5% CO2 incubator for 14 d, then each well was cleaned twice with PBS, dried in air, fixed by methanol for 15 min, stained with Giemsa for 15 min, and dye was washed away with running water, dried again in air, finally clone was counted more than 50 cells under microscope, per clone formation rate of cells was calculated by following formula: clone formation rate of cells (%) = (clone number/inoculated cell number) × 100%; relative clone formation rate of cells (%) = (clone formation rate of radiation group/clone formation rate of control group) × 100%Hela/TK cells were passaged and harvested by standard trypsin digestion, single cell was suspending in inoculum. Percentage of single cell was over 95%, 1060Co radiotherapy group and combined treatment group, 10 mice in each group. Each nude mouse was injected sc on both sides of flanks with 3.0 × 106 Hela/TK cells. When tumor grew to 1 cm in diameter, treatment as following: ① gene treatment group: GCV (100 mg/kg) was given intra-peritoneal cavity (i.p), injection volume 0.4 ml/mouse, once one day, continuous injection for 14 d; ② 60Co radiotherapy group: Synchronous with gene treatment group, tumor was radiated with 60Co once, absorption dose was 20 Gy; ③ combined treatment group: GCV (100 mg/kg) was given i.p, 2 h later, tumor tissue radiated with 60Co once, absorption dose was 20 Gy. Other steps were similar to gene treatment group: ④ Control group: same volume of aseptic phosphate buffer solution was given i.p, but without 60Co radiation. Four weeks later mice were sacrificed, tumor was resected and then their weight was weighed respectively with electro-balance.Forty BALB/c nude mice were divided into 4 groups: control group, gene treatment group, Tumor inhibition rate (%) was calculated as /average tumor weight of control group × 100%.60Co radiotherapy group and combined treatment group).Radiosensitization was determined by E/O value  which was reported by Yunfei Xia et al  whether Experimental data were presented as means ± the standard deviations (SD) of four independent experiments performed in triplicate. Where appropriate, samples were analyzed by using the Student's two-tailed t-test or ANOVA by SPSS software. And statistical significance was defined as P < 0.05 and P < 0.01.th day, inhibition rates of Hela/TK cell growth in the three treatment group were 45.8 ± 7.3%, 42.4 ± 5.6% and 87.5 ± 10.32% respectively, compared with gene treatment group and radiotherapy group, there was a significant difference in combined treatment group (P < 0.01) .The percentages of Hela/TK cells apoptosis were analyzed by FCMs on the fluorescence channel as described by Zhang . The resP > 0.05), compared with that of control group. whereas there was a significant difference between clone formation rate of simple radiotherapy group and clone formation rate of combined treatment group, compared with that of control group, their relative clone formation rates were 51% and 26% respectively  × (T3/T1) = 0.38; 0 = (T4/T1) = 0.12; E/0 = 3.17 (> 1.4). Above the results indicated the radiosensitization of HSV-TK/GCV suicide gene system was existing(E/O = 3.17 > 1.4).E = . The ratio of apoptosis among the gene treatment group, radiotherapy group and combined treatment group were (22.76 ± 3.51)%, (25.16 ± 3.17)%, (45.27 ± 5.15)% respectively compared with gene treatment group and radiotherapy group, difference in combined treatment group was significant (P < 0.01). Moreover, In vivo nude mice cervical cancer model treated experiment showed that there were significant differences in the three treatment groups, compared with control group; there was a significant difference in combined treatment group(P < 0.01), compared with gene treatment group or radiotherapy group, combined treatment group was superior to simple treatment group, which was in consistent with the study of NishiharaI et al [60Co may have synergism, when GCV less than effective dose was used in combined 60Co radiotherapy, sensitivity of HSV-TK gene to rays was enhanced significantly.Tumor gene-radiotherapy is a new thought put forward in recent years to treat tumor. It transfers gene into the body, which can kill tumor and enhance sensitivity of radiation. It has synergism of radiation and gene in local radiotherapy . Recent 51)%, 25. ± 3.17%,51)%, 25. ± 3.17%,With regard to mechanism of the radiosensitization of HSV-TK/GCV suicide gene system, it was believed that when GCV enters cells transfected by HSV-TK gene, it converts into GCV-triphosphate, which can inhibit tumor cell repair of potentially lethal damage (PLD) and mix in DNA as competitive inhibitor of dTTP to enhance radiation sensitivity, reduce dose and toxic effect and improve therapeutic effect . At presOn the whole, there were intimate relation between HSV-TK gene and tumor radiosensitivity. With molecule radiobiological development, it will be more clear that HSV-TK can enhance sensitivity of radiation and radiotherapy can enhance effect of suicide gene to inhibit tumor, which will give malignant tumor treatment more new approach and expectation; Meanwhile it will be very important to formulate correct combined therapeutic regimen and to predict curative effect and to appraise prognosis.The authors declare that they have no competing interests.vivo animals experiments. DC carried out the in vitro experiments including growth inhibition experiment of cells and clone formation rate of cells experiment and apoptosis assay with flow cytometry, etc. and participated in the design of the study and performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.QT conceived of the study, and participated in its design and coordination, and carried out in The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/609/prepub


Hyperproinsulinaemia is associated with obesity and is a risk factor for Type 2 diabetes. We explored the dynamics of proinsulin and insulin and postprandial effects on glucose and lipids in subjects who had undergone gastric bypass (GBP) surgery compared with morbidly obese (MO) subjects and normal weight control subjects (NW).sd, 34.8±6.2kg/m2] who had undergone GBP surgery, 10 MO subjects (BMI 44±3.1kg/m2) and 12 NW control subjects (BMI 23.2±2.4kg/m2). After an overnight fast, a standard meal (2400 kJ) was ingested and glucose, proinsulin, insulin free fatty acids and triglycerides were determined up to 180 min.Subjects free from diabetes were recruited: 10 previously MO subjects [body mass index (BMI)±P < 0.05). Postprandial AUC for glucose was similar in the three groups and AUC for proinsulin was high in MO, intermediate in the GBP group and lowest in NW control subjects (P for trend = 0.020). Postprandial proinsulin at 60 min was similar in the GBP group and MO subjects and twofold higher than in NW control subjects. Postprandial proinsulin at 180 min was normal in the GBP group, but fivefold increased in MO subjects (P = 0.008). Insulin increased rapidly at 30 min in the GBP group and was normal at 90 min, whereas insulin was still increased at 90–180 min in the MO subjects (P < 0.001).Fasting proinsulin was similar in the GBP group and NW control subjects, but threefold increased in MO subjects (MO subjects, free from diabetes, have elevated proinsulin concentrations in the fasting as well as the postprandial phase. After GBP surgery markedly lower fasting and postprandial proinsulin concentrations were observed, although BMI was higher compared with NW control subjects. Obesity is associated with insulin resistance , fastingPatients were recruited from the out-patient clinic for obesity care, University Hospital, Uppsala, Sweden, where preassessments before and long-term check-up visits of patients after surgery were performed. Control subjects were recruited locally. All subjects were White. Exclusion criteria for all three groups were established diabetes or use of glucose-lowering agents, as well as use of ACE-inhibitors, angiotensin II receptor blockers or β-blockers, to avoid possible influence on glucose, proinsulin and insulin status.Three groups of subjects were compared2 (mean ±sd), who had undergone GBP surgery 1–5 years (mean 4.1 years) before study. The mean presurgery BMI was 45.3 ± 7.6 kg/m2. The mean absolute weight loss was 31.8 kg (range 16–47 kg). The mean fall in BMI after surgery was 10.5 kg/m2 (23%).Ten surgically treated patients considered weight stable after weight loss due to GBP surgery, with body mass index (BMI) 34.8 ± 6.2 kg/m2. These patients had refused surgery or were on the waiting list for surgery after preassessment. Their mean BMI was similar to the presurgery BMI in the group of subjects who underwent GBP surgery (P for difference = 0.59).Ten surgically untreated patients with MO, with BMI 44.0 ± 3.1 kg/m2. Age and sex distribution were similar in the three groups , consisting of: carbohydrates 68.2 g, fat 22.3 g, proteins 24.6 g and fibre 6.4 g.After an overnight fast for 17 h the standardized test meal was ingested at 13.00 h in the obesity out-patient clinic, allowing supervision of food intake, which was finished within 15 min. Blood samples were collected, centrifuged and freshly frozen immediately before the test meal and thereafter at 30, 60, 90, 120 and 180 min after ingestion. Data were collected on preprinted Case Report Forms.2) was calculated. All patients completed a standardized questionnaire, including questions concerning their health status and smoking habits.Weight (kg) and height (m) were measured on standardized, calibrated scales and BMI  on a Bio-Rad Coda automated EIA analyser . Concentrations of serum FFA were determined using the Wako NEFA C-test kit  and serum TG by a lipase and quinoneimine dye method (Konelab) on a Konelab analyser . At the Department of Medical Sciences, University Hospital in Uppsala, plasma glucose concentrations were determined using a routine glucose oxidase technique .All plasma samples before and after the meal for all subjects were analysed as one batch, thus avoiding drift of determinations.1c, aspartate aminotransferase, alanine aminotransferase, creatinine, sodium, potassium, haemoglobin and white cell count were analysed using routine methods at the Department of Clinical Chemistry, University Hospital, Uppsala.Basal routine tests for concentrations of HbAa priori. Results are given as arithmetic mean with sd and sem. anova and Students t-test were used for group comparisons. Tests were two-tailed and a P-value < 0.05 was considered significant. Test meal data are given as absolute concentrations  in the GBP group and 0.5 mmol/l higher in the MO group compared with the NW control subjects. HbA1c was 0.3% higher in both the GBP and MO groups compared with NW control subjects (P < 0.05 for both).Clinical characteristics of the study subjects are presented in Fasting proinsulin and insulin did not differ between the GBP and NW control subjects, whereas proinsulin and insulin concentrations were significantly higher in the MO group compared with the GBP and NW control subjects, respectively. The mean fasting FFA value in the MO and GBP groups was not significantly different. TGs were higher in MO group, as expected.Absolute concentrations of glucose, proinsulin, insulin, FFA and TG are shown in sd) for glucose (mmol × min) did not differ between the MO group (270 ± 92), the GBP group (310 ± 110) and NW control subjects (250 ± 130) (P for trend = 0.27).The AUC (mean ±Data for changes in glucose are presented in sd) for proinsulin (pmol × min) was largest in the MO group (7600 ± 5300), intermediate in the GBP group (5300 ± 2700) and lowest in NW control subjects (3000 ± 1600) (P for trend = 0.02). Data for changes in proinsulin are shown in The AUC (mean ±sd) for insulin (pmol × min) was highest in the MO group (41 000 ± 9900), intermediate in the GBP group (32 000 ± 16 500) and lowest in NW control subjects (18 000 ± 8300) (P for trend < 0.001). Data for changes in insulin are presented in The AUC (mean ±P = 0.48 and 0.39).The proinsulin to insulin ratio decreased identically in all three groups during the first 30 min. The mean ratio decreased from approximately 0.32–0.10, with no significant difference between groups (sd) for FFA (mmol × min) did not differ between the MO group (84 ± 31), the GBP group (68 ± 24) and NW control subjects (63 ± 54) (P for trend = 0.44).The AUC (mean ±P = 0.03–0.007) at each postprandial time point except at 180 min in the MO group compared with the GBP group and NW control subjects. Prior to 180 min, the FFA values were all lower in the GBP group compared with the NW control subjects and the MO group.FFA absolute concentrations, shown in No significant differences were observed regarding postprandial changes in circulating FFA and concentrations between the three groups, except at 180 min, where FFA were higher in the GBP group .sd) for TG (mmol × min) was greatest in the MO group (32 ± 29), intermediate in the GBP group (16 ± 22) and lowest in NW control subjects (8 ± 15), although the differences did not reach statistical significance (P for trend = 0.07). TG absolute concentrations, presented in P = 0.06–0.003) at each postprandial time point in the MO group compared with the GBP group and NW control subjects. No significant differences were observed regarding postprandial changes in circulating TG and concentrations between the three groups, except at 180 min, where TG were higher in the MO group  and with AUC for insulin , but not with AUC for glucose , AUC for FFA  or AUC for TG .BMI correlated with AUC for proinsulin  and T2DM . FastingThe relative hypoinsulinaemia in the late phase of the test in the GBP group followed the rapid decrease in both glucose and insulin after peak concentrations in the early phase, and coincided with the time point when symptoms of the so-called dumping syndrome may occur . The mecIn summary, GBP surgery results in dramatically improved insulin sensitivity and rapid meal-stimulated secretions of proinsulin and insulin with sustained effects on glucose and lipid metabolism. Our results suggest that future studies should determine whether proinsulin is a marker for diabetes risk before and after GBP surgery. It might be speculated that the proinsulin status can be used as an indication for bariatric surgery. To answer such questions, further research on proinsulin and insulin as markers for T2DM and CHD in long-term follow-up after GBP surgery is warranted.None to declare.


Noccaea caerulescens acclimation to Cd exposure by elucidating the process of photosystem II (PSII) acclimation by chlorophyll fluorescence imaging analysis. Seeds from the metallophyte N. caerulescens were grown in hydroponic culture for 12 weeks before exposure to 40 and 120 μM Cd for 3 and 4 days. At the beginning of exposure to 40 μM Cd, we observed a spatial leaf heterogeneity of decreased PSII photochemistry, that later recovered completely. This acclimation was achieved possibly through the reduced plastoquinone (PQ) pool signaling. Exposure to 120 μM Cd under the growth light did not affect PSII photochemistry, while under high light due to a photoprotective mechanism (regulated heat dissipation for protection) that down-regulated PSII quantum yield, the quantum yield of non-regulated energy loss in PSII (ΦNO) decreased even more than control values. Thus, N. caerulescens plants exposed to 120 μM Cd for 4 days exhibited lower reactive oxygen species (ROS) production as singlet oxygen (1O2). The response of N. caerulescens to Cd exposure fits the ‘Threshold for Tolerance Model’, with a lag time of 4 d and a threshold concentration of 40 μM Cd required for the induction of the acclimation mechanism.We provide new data on the mechanism of  Cadmium is a non-essential heavy metal that can occur in the environment in high concentrations as a consequence of numerous human activities, thus becoming toxic to all organisms ,2,3,4,5.Hyperaccumulators are plant species that vigorously take up heavy metals, translocate them into the above-ground parts and isolate them into a risk-free state ,11. ThesNoccaea caerulescens is known as a zinc–cadmium–nickel hyperaccumulator because it can accumulate these metals at extremely high concentrations in its aboveground tissues [N. caerulescens can store as much as 14,000 μg Cd g−1 dry biomass without showing toxicity signs [ tissues , and has tissues . It has  tissues ,21. Certty signs ,23,24. Cty signs .2 fixation [Photosynthesis has been shown to be very sensitive to Cd either directly or indirectly ,29,30,31fixation ,35, or ffixation ,37. Photfixation ,33,38,39fixation ,40.Noccaea caerulescens. Our previous study indicated that despite the substantial high toxicity levels of Zn and Cd in N. caerulescens aboveground tissues, the photochemical energy use at PSII did not differ compared to controls [N. caerulescens acclimation to Cd exposure and to clarify the process of photosynthetic acclimation, we treated in hydroponic culture N. caerulescens plants with 40 and 120 μM Cd for 3 and 4 days.We investigated photosynthetic acclimation to Cd toxicity using the hyperaccumulator controls . HoweverNoccaea caerulescens F.K. Mey collected from a former Copper Mine area at Røros (Norway) were cultivated hydroponically in an environmental growth chamber as described previously [Seeds of eviously .4.8H2O) for 3 and 4 days while others were left to control growth conditions.After growth for 12 weeks, some plants were exposed to 40 or 120 μM Cd , 40 or 120 μM Cd for 3 and 4 days, as described previously [−2 s−1, GL) and a high light intensity . The measured and calculated chlorophyll fluorescence parameters with their definitions are given in Chlorophyll fluorescence measurements were carried out with an Imaging-PAM Chlorophyll Fluorometer  in dark-adapted leaves (15 min) of eviously ,41. FiveRepresentative results of the measured chlorophyll fluorescence parameters are also displayed as color-coded images.t-test (p < 0.05). Five leaves from five different plants were analyzed in each treatment. In all graphs, the error bars are standard deviations, while columns with the same letter are not statistically different at p < 0.05.All measurements that are expressed as mean ± SD were analyzed by student Fv/Fm) in N. caerulescens decreased significantly but increased to control values at 120 μM Cd , at both growth light (GL) and high light (HL) intensity decreased significantly compared to the control, after 3 d at 40 μM Cd, while it improved during the 4 d  decreased significantly compared to the control after 3 d at 40 μM Cd at GL and increased to control values during the 4 d , a loss process due to PSII inactivity, at both GL and HL intensity, increased significantly compared to the control after 3 d exposure to 40 μM Cd, while during the 4 d it decreased compared to 3 d  that reflects heat dissipation of excitation energy, decreased significantly compared to the control after 3 d at 40 μM Cd at GL, while it improved during the 4 d a. ExposuThe electron transport rate (ETR), at both GL and HL intensity, decreased significantly compared to the control after 3 d at 40 μM Cd, while it improved during the 4 d . HoweverqP) that is a measure of the fraction of open PSII reaction centers, at both GL and HL intensity, decreased significantly compared to the control after 3 d at 40 μM Cd, while it improved during the 4 d  in ox state d.Fv/Fm) show the smallest spatial heterogeneity even though it decreased significantly at 40 μM Cd and increased to control values at 120 μM Cd (PSII) decreased significantly after 3 d at 40 μM Cd at GL, while it improved during the 4 d, showing a high spatiotemporal leaf heterogeneity (NO) (qP) (NO) increased; thus, these areas exhibited increased singlet oxygen (1O2) production  (NO (qP (The maximum quantum efficiency of PSII photochemistry (20 μM Cd a. The quogeneity b. Among ity (NO) d and theNO) (qP) e. The moNO) (qP) d,e. At toduction d, and alP values e. Howeveecreased d and thesed (qP) e. At exp(qP)  (NO) (Exposure of ogeneity  and the ity (NPQ b that doQ (PSII) a and decII) (NO) c.2+ of the Mg2+ ion in the chlorophyll molecules of the light-harvesting complex II, while in HL it is mainly from direct damage to the PSII reaction center [The type of damage on PSII that has frequently been identified as the main target of Cd toxicity on photosynthesis strongly depends on light conditions ,44,45,46N. caerulescens leaves grown under control growth conditions at both GL and HL show a spatial heterogeneity in PSII functionality (s) [PSII) arises from local differences in internal CO2 concentrations, which in turn result from changes in stomatal conductance due to patchy stomatal behavior [PSII can be used to calculate stomatal conductance [ionality . This splity (s) ,48. Stomlity (s) ,50. It ibehavior . A body ductance ,53,54,55PSII decreased significantly at the left and right leaf sides (NPQ (NO)  via the triplet state of chlorophyll (3chl *) [N. caerulescens leaves exhibited increased 1O2 production at the left and right leaf sides, since ΦNO increased significantly at those areas. Thus, although Cd2+ is a redox-inert element, it produces reactive oxygen species [2O2) [At the beginning of exposure to 40 μM, Cd Φaf sides a, with ades (NPQ b resultiNPQ (NO) c. The int energy ,57. ΦNO (3chl *) ,58,59. A species . The simes [2O2) . The stoPSII increased at the left and right leaf sides  decreased even more than control values, and thus exhibited lower singlet oxygen (1O2) production. This was due to the photoprotective mechanism that can divert absorbed light to other processes such as thermal dissipation, preventing the photosynthetic apparatus from oxidative damage [During the 4 d exposure to 40 μM Cd, Φaf sides a, with ades  in N. caerulescens leaves exposed to 40 μM Cd for 3 d  production corresponds to spatial accumulation metal patterns [It seems that spatiotemporal variations in the redox state of the PQ pool related to stomatal conductance, an indicator of the extent of stomatal opening , are intpatterns . A hetermulation . Still, patterns .N. caerulescens to Cd exposure fits the ‘Threshold for Tolerance Model’, with a lag time or/and a threshold concentration required for the induction of a tolerance mechanism [N. caerulescens to activate stress-coping mechanisms.In our work, the response of echanism ,76,77,78echanism . In accoN. caerulescens to Cd exposure fits the ‘Threshold for Tolerance Model’, with a lag time of 4 d and a threshold concentration of 40 μM Cd required for the induction of the acclimation mechanism through the reduced PQ pool that mediated stomatal closure probably by the generation of mesophyll chloroplastic hydrogen peroxide (H2O2) [Acclimation to Cd exposure was achieved through the possible Cd detoxification mechanism done by vacuolar sequestration and the reduced plastoquinone (PQ) pool signaling that mediated stomatal closure and decreased Cd supply at the affected leaf area. The response of e (H2O2) , which ae (H2O2) ,80, as we (H2O2) ,62,63. Te (H2O2) ,44,45,46e (H2O2) ,82,83, pe (H2O2) . As it w


The characteristic fragmentation pattern of alkaloids, phenolic acids, tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides, 2-(2-phenylethyl) chromones, phthalides and gingerol-related compounds were tentatively elucidated using structurally-relevant product ions. It was observed that neutral losses of C9H10O3 and C9H8O2 were the characteristic product ions of scopola alkaloids. Neutral fragment mandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge, tropylium ion and C4H2O unit were the characteristic ions of 2-(2-phenylethyl) chromone, which resulted from the Retro-Diels-Alder (RDA) cleavage of the C ring. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in TCM. This work is expected to provide comprehensive information for the quality evaluation and pharmacokinetic studies of JTT.In the present study a universally applicable HPLC-DAD/ESI-MS/MS method was developed for carrying out the comprehensive characterization of Jitai tablets (JTT). Based on the ESI-MS Rhizoma Corydalis, Radix Salviae Miltiorrhiae, Radix Angelicae sinensis, Rhizoma Chuanxiong, Semen Persicae, Flos Carthami, Radix Aconite, Radix Ginseng, Cortex Cinnamomi, Rhizoma Zingiberis, Semen Myristicae, Flos Daturae, Radix Aucklandiae, Lignum Aquilariae Resinatrm and Margarita. JTT have been proved to be very safe and effective in the inhibition of protracted withdrawal symptoms with less harmful side effects +, [M+H−NH4]+ and [M+Na]+ were observed in positive ion ESI mode. The information of [M−H]−, [M+H]+ and [M+Na]+ were used to determine molecular weight. The identification of these compounds was carried out referring to their MS and MSn spectra, and the characteristic fragmentation pattern of alkaloids, phenolic acids, tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides, 2-(2-phenylethyl) chromones, phthalides and gingerol-related compounds are summarized as follows. In the full scan mass spectra, [M−H]Maximum absorptions in the range from 260 to 300 nm are exhibited by alkaloids in their UV spectra, and the preliminary identification could be facilitated by this characteristic absorption . Consequ+ ions of tertiary alkaloids in ESI-MS/MS spectra were observed to undergo the Retro-Diels-Alder (RDA) cleavage. The fragmentation pathway is shown in Using the proposed method, 10 isoquinoline alkoloids including four tertiary alkaloids, and six quaternary alkaloids were identified. In the positive ionization mode, the protonated molecule [M+H]3]+ with characteristic m/z value were exhibited by quaternary alkaloids. In addition, fragment ions [M−CO−CH3]+ and [M−2CH3]+ were observed in the MS/MS spectra, which indicated unique fragments produced by the losses of CO and CH3. By using structurally-relevant product ions in literature data + and [M+H−H2O−2CH3OH]+ were exhibited in their MS/MSspectra, which indicated a further fragmentation by the neutral losses of CH3OH eliminated from the methoxyl on C16 position and the proton on the C15 position. By using structurally-relevant product ions in literature data + was formed by the loss of tropic acid, while m/z 156 [M+H−C9H8O2]+ was produced by the loss of C9H8O2 unit resulted from the fragmentation reaction of the ester bond. It could be concluded that the ions at m/z 138 and 156 were the characteristic product ions of scopola alkaloids. By comparison with reference standards and literature data ure data , the − ion and [M−H−C9H8O4]− ion were obtained via dissociation of the bonds on either side of the esterifiable carboxyl oxygen in polymers, which also showed the same general fragmentation pattern for their quasi-molecular ions + ion of peak 100 was observed in MS spectra, and m/z 277 [M+H−H2O]+ ion was exhibited in MS/MS spectra, which corresponded to two fragment ions at m/z 280[M+H−CH3]+ and m/z 262 [M+H−H2O−CH3]+, indicating the loss of H2O and CH3 in the fragmentation pattern. Therefore, tanshinone IIA was unambiguously identified by comparison with the reference standard. Similarly, the [M+H]+ ions of cryptotanshinone and isotanshinone IIA, eluting at 75.1 and 76.4 min in ESI-MS/MS spectra, were identified respectively.In the positive ionization mode, the protonated molecule [M+H] spectra . For exaFlos Carthami. In the full scan mass spectra, most ions were selected to be dissociated with CID in the MS/MS spectra. Although flavonoid glycosides can be ionized under both positive and negative ionization conditions, more relative intense signals were obtained in negative ionization mode. Usually, high abundant quasi-molecular [M−H]− ion was observed in the MS spectra. In the MS/MS spectra, aglycone ions m/z 285, m/z 301 and m/z 287 were finally formed by losing several glycosidic units, which were identified as the characteristic ions of kaempferol, quercetin/hydroxy kaempferol and chalcone, respectively − were firstly exhibited by these two compounds. In the MS/MS spectra, [M−H−C8H7NO]− ions were finally formed by loss of neutral fragments mandelonitrile, which was also the characteristic ion of amygdalin and prunasin. In addition, a [M−H−Glc−C8H5N]− fragment was also observed in MS/MS spectra, according to the proposed fragmentation pathway shown in The UV spectra of peaks 10 and 16 showed maximum absorption bands at 210–230 nm, consistent with the characteristic absorption of cyanogenic glycosides reported in Persicae . In the − and [M−H+HCOOH]− were formed first. In the MS/MS spectra, aglycone ions at m/z 475 and 459 were finally formed by losing several glycosidic units, which were also the characteristic ions of panaxatriols and panaxadiols, respectively. Usually, the ginsenosides could be quickly identified by their big molecular weight (>600 Da). However, the comparison with reference standards should also be needed due to the similarity of the structures of some ginsenosides [m/z 1078 [M−H]− in MS spectra, m/z 945 [M−H−(Araf/Xyl)]−, m/z 783[M−H−(Araf/Xyl)−Glc]−, m/z 621 [M−H−(Araf/Xyl)−2Glc]− and m/z 459 [Agl]− ions could be detected in their MS/MS spectra, which exhibited a fragmentation pathway corresponding to the loss of glycosidic units. The fragment ion at m/z 459 corresponding to the protopanaxadiol aglycon moiety was a characteristic ion to identify the structures of these two compounds [m/z 955[M−H]− in MS spectra, and exhibited m/z 793 [M−H−Glc]−, m/z 613 [M−H−2Glc−O]− and m/z 455 [Agl]− ions in the MS/MS spectra. The ion at m/z 455 was produced by successive losing of all linked glucosidic bonds, which was a characterized fragmentation of oleanolic acid type ginsenoside. By comparison with the literature data, the compound was tentatively identified as ginsenoside Ro enosides . For exaompounds . By composide Ro . With thThe UV spectra of peaks 18, 22, 25, 80, 84 and 86 showed two absorption maximum at about 230 nm and 280 nm, consistent with the characteristic absorption of 2-(2-phenylethyl) chromones . − of peaks 80, 84 and 86 were observed in negative ion mode, and [M+H]+ and [M+H−H2O]+ in positive ion mode. In the MS/MS spectra, high abundant product ions at m/z [M+H−C7H7]+ were formed by the loss of tropylium ions resulted from the RDA cleavage of C ring, while m/z [M+H−C7H7−C4H2O]+ ions were produced by the cleavage of B ring. It could be concluded that C7H7+ and C4H2O units were the characteristic losses of 2-(2-phenylethyl) chromones in both negative and positive ion mode. By comparison with literature data ure data , peaks 82O and carbonyl group + in MS spectra, and exhibited fragmental ions at m/z 189[M+H−2H2O]+, m/z 179[M+H−H2O−CO]+, m/z 165[M+H−H2O−CO−CH2]+ and m/z 145[M+H−2H2O−CO2]+ in MS/MS spectra. According to the law that the trans-production peaks eluted early than cis peaks in reversed-phase column and comparing with the literature data − in negative mode, but also m/z [M+H−H2O]+ in positive mode, and the fragment ions of both were differentiated by units of 28 Da (C2H4), which suggested that they were homologues with different alkyl chain length [m/z 293 [M−H]− and high abundant ionat m/z 277 [M+H−H2O]+ in MS spectra. In the MS/MS spectra, m/z 177 [M+H−H2O−C6H12O]+ and m/z 145 [M+H−H2O−C6H12O−CH4O]+ were exhibited, which indicated a further fragmentation by the loss of a neutral alkyl moiety and a rearrangement. By comparison with literature data + of peaks 89 and 101 were formed in the MS/MS spectra, which indicated the characteristic fragmentation of the keto group on the alkyl chain. By comparison with the literature data  chromone was isolated and purified from JTT and each comprised drug were ground into a fine powder and accurately weighed (400 mg) in a vial. 50% Methanol solvent (4.0 mL) was added and ultrasonic extracted for 30 min to prepare a uniform suspension, then centrifuged at 13,680 g for 10 min . At last, the supernatant was filtered using a syringe filter (0.22 µm).All reference standards were accurately weighed, and dissolved in methanol to obtain stock solutions with proper concentrations . All the stock solutions were stored in the refrigerator at 4 °C until analysis.+) and 10,000 (ESI−) and scan range 100–1,500 amu. The amplitude voltage used for fragmentation was 1.0 V. The capillary exit voltage was set at 121 V for both positive and negative ion modes. Positive and negative mode data were acquired using Agilent chemstation software .HPLC/DAD analysis was carried out on an Agilent 1100 series HPLC system  equipped with a quaternary pump with on-line degasser, auto-sampler, column oven and diode array detector (DAD), scanning from 200–400 nm and the wavelength was then selected and fixed at 280 nm since many peaks could only be found under this condition. Chromatographic separation was performed on a XTerra MS C18 column  equipped with an XTerra MS C18 guard column  (Waters), with the column temperature was set at 25 °C. The mobile phase consisted of 0.1% aqueous formic acid (A) and (B) acetonitrile using a gradient elution of 5–30% B at 0–50 min, 30–100% B at 50–90 min, 100% B at 90–100 min. The flow rate was kept at 1.0 mL/min and 10 µL of sample solution was injected in each run. By solvent splitting, 0.2 mL/min portions of the column effluent were delivered into the ion source of an Agilent LC-MSD Trap XCT mass spectrometer. The MS conditions were fixed as follows: drying gas (nitrogen) flow rate 10 L/min, gas temperature 350 °C, pressure of nebulizer gas 30 psi, HV voltage 3.5 kV, compound stability 100%, threshold 50,000 . An in-house library was established by searching from online websites such as Google Scholar, PubMed of the US National Library Medicine and Chinese National Knowledge Infrastructure (CNKI) of Tsinghua University, all components of comprised drugs were summarized in a Microsoft Office Excel sheet, which included the name, molecular weight, deprotonated molecular ions, protonated molecular ions, fragment ions and literatures of each published known compound. The “Find” function of Microsoft Office Excel was applied to match the detected mass value with that of published known compounds in the library. The characteristic fragmentation pattern of all components was tentatively elucidated using structurally-relevant product ions.In the present study, a reliable and combinative analytical method using HPLC-DAD/ESI-MS/MS was established for rapid identification of multiple constituents in JTT. As a result, a total of 101 constituents were successfully separated and identified, and the fragmentation patterns of nine types of natural products in the complex system were successfully elucidated by the proposed method. n) method for screening the chemical constituents in such complex formula comprised of fifteen drugs has been developed. Furthermore, according to the interpretation of their mass behavior obtained from HPLC-DAD/ESI-MS/MS spectra and also taking into account the data provided by the 23 reference standards and the established in-house library, a total of 101 constituents were systematically characterized and identified in a single run. Neutral losses of C9H10O3 and C9H8O2 were the characteristic product ions of scopola alkaloids, and neutral fragment mandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge, losses of tropylium ions and C4H2O unit resulted from the RDA cleavage of C ring existed in fragmentation of 2-(2-phenylethyl) chromones. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in TCMs. This work is expected to provide comprehensive information for the quality evaluation and pharmacokinetic studies of JTT.The present study, compared with the previous studies, showed differences or improvements as follows. First of all, it is the first time a combinative LC/MS (MS


Astrocytes are the most widely distributed cells in the brain, and astrocyte apoptosis may play an important role in the pathogenesis of neurodegenerative diseases. Folate is required for the normal development of the nervous system, but its effect on astrocyte apoptosis is unclear. In this study, we hypothesized that folic acid (the therapeutic form of folate) decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. Primary cultures of astrocytes were incubated for 12 days with various concentrations of folic acid (0–40 μmol/L), then cell proliferation, apoptosis, intracellular folate concentration, intracellular homocysteine (Hcy) concentration, intracellular reactive oxygen species (ROS) levels, telomeric DNA oxidative damage, and telomere length were determined. The results showed that folic acid deficiency decreased intracellular folate, cell proliferation, and telomere length, whereas it increased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In contrast, folic acid dose-dependently increased intracellular folate, cell proliferation, and telomere length but it decreased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In conclusion, folic acid inhibited apoptosis in astrocytes. The underlying mechanism for this protective effect may be that folic acid decreased oxidative stress and thereby prevented telomeric DNA oxidative damage and telomere attrition. Telomeres are ribonucleoprotein structures capping the end of every linear chromosome . They arFolate (vitamin B9) acts as a coenzyme to transfer one-carbon units that are necessary for deoxythymidylate synthesis, purine synthesis, and various methylation reactions. For example, folate is an important co-factor in the re-methylation process of homocysteine (Hcy) metabolism . ConsequFolic acid is the form of folate that is used therapeutically. It stimulates the proliferation of neural stem cells and the differentiation of neurons in vitro and in vivo ,18. Folir = 0.756, p = 0.004). Cell proliferation rate in the high-dose folic acid group (40 μmol/L) was 1.1 times that in the normal folic acid group , since the total percent apoptosis rates in the presence of folic acid at 0, 10, 20, or 40 μmol/L were, respectively, 14.33 ± 4.2, 6.42 ± 3.00, 5.15 ± 2.79, 3.78 ± 3.10 , since the total percent apoptosis rates in the presence of folic acid at 0, 10, 20 or 40 μmol/L were, respectively, 16.73 ± 2.03, 7.64 ± 0.53, 6.57 ± 0.76, 5.06 ±0.91 (Incubation for 12 days with various concentration of folic acid (0–40 μmol/L) had multiple effects on astrocytes. Folic acid deficiency decreased cell proliferation compared with high-dose folic acid treatments (20 or 40 μmol/L) a. Folic 8 ± 3.10 a,c. Hoec06 ±0.91 b,d. Takep < 0.05)  increased intracellular folate compared with the normal dose of folic acid (10 μmol/L) ( < 0.05) a. The in < 0.05) b. Simila < 0.05) c.r = −0.907, p = 0.000)  (The incorporation ratio of 8-oxoG within telomeric DNA was increased by folic acid deficiency and decreased by high folic acid concentrations (20 and 40 μM) in a dose-dependent manner (= 0.000) . The rat= 0.000) .r = 0.885, p = 0.000)  c.The present study determined that folic acid deficiency in primary astrocytes decreased intracellular folate, telomere length, and cell proliferation but increased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In contrast, incubation with folic acid for 12 days dose-dependently increased cell proliferation and telomere length. Furthermore, folic acid dose-dependently decreased Hcy, ROS, telomeric DNA oxidative damage, and apoptosis. Previous studies reported that folate deficiency is associated with apoptosis in neural stem cells and neurons ,22. The Folate deficiency inhibits the re-methylation process of Hcy metabolism and thereby causes Hcy to accumulate . This meA previous study in primary astrocytes found that exposure to elevated Hcy concentrations caused endoplasmic reticulum stress and impaired autophagy that were reversed by supplementation of the cell culture medium with folate and vitamin B12 . AnotherTelomeres cap chromosome ends and are essential for genome stability, cell proliferation, and human health . With eaIn this study, 10 μmol/L of folic acid was used as the normal concentration. Dulbecco’s modified eagle medium (DMEM) has been recommended for the culture of primary astrocytes and it is also the traditional medium for the culture of neuronal cells. Since DMEM medium contains folic acid at 4 mg/mL (ca. 10 μmol/L) as a standard constituent, we chose 10 μmol/L folic acid as the normal concentration. However, the level used in this study is higher than the physiological level. Further study is needed to discuss the effect of folic acid at the physiological level in vivo.This study has a limitation. This study hypothesized a mechanistic link between folate protection against astrocyte apoptosis and reduced telomere attrition and oxidative stress. The mechanism of oxidative stress-induced telomere attrition is only one of the potential mechanisms of folate protection. Further experiments are needed to demonstrate our hypothesis. However, astrocytes are the most widely distributed cells in the brain, and astrocyte apoptosis may contribute to the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease. Also, folate metabolism is associated with the survival of astrocytes in the brain. The anti-apoptotic effect of folic acid on astrocytes may be exploited to improve neurodegenerative disorders prevention and therapeutic treatment.3 pieces and digested with 0.25% trypsin at 37 °C for 15 min. Cells were seeded in DMEM  containing 10% fetal bovine serum (FBS) , 10 μM folic acid , and 1000 mg/mL glucose. The cells were cultured in 95% air and 5% CO2 at 37 °C, and the culture medium was changed every 2 or 3 days. After 7 days, the oligodendrocytes and microglia were removed by thermostatic oscillation at 200 rpm for 18 h. Astrocytes were identified by immunofluorescence with an antibody against glial fibrillary acidic protein (GFAP) . Cultures that were >95% GFAP-positive were used for the experiments . Primary astrocytes were prepared as described previously . In brieeriments . These p® AQueous One Solution Cell Proliferation assay . Cells were seeded at a density of 5000 cells/per well with 100 μL of culture medium in 96-well plates, then 20 μL CellTiter 96® AQueous One Solution Reagent was pipetted into each well, and the plates were incubated for 4 h at 37 °C in a 5% CO2 incubator. Absorbance was recorded at 490 nm using a microplate reader .Cell proliferation was measured with the CellTiter 966 cells were collected, washed twice with D-PBS , resuspended in 1×binding buffer, and incubated with 5 μL AnnexinV–FITC and 5 μL propidium iodide (PI) at room temperature for 20 min, followed by the addition of 400 μL of 1× binding buffer. The cells were sorted by a flow cytometer , and the data were analyzed with Flowjo 7.6. Nuclear morphology was detected by fluorescence staining using Hoechst 33342, and the data were analyzed with Image Pro Plus 6.0. Apoptotic cells were defined on the basis of their nuclear morphology, such as chromatin condensation and fragmentation. Cell apoptosis was measured by flow cytometry and fluorescence staining with Hoechst 33342. Briefly, 1 × 10® 2000 Folic Acid kit and an IMMULITE®2000 System Analyzer , according to the manufacturer’s instructions. The IMMULITE 2000 performs a 2-cycle, on-board sample treatment based on the principle of competitive immunoassay, which can detect folate, dihydrofolate, and tetrahydrofolate. Hcy concentration was measured with an ELISA kit  in accordance with the manufacturer’s instructions. Microplates that had been precoated with antibodies specific for Hcy were read at a wavelength of 490 nm by a microplate reader . The standard curve was generated using Curve Expert 1.3 software . The intracellular concentrations of folate and Hcy were normalized to cell protein content, which was measured using a BCA total protein quantitative assay kit .Intracellular folate concentration was determined with the IMMULITE2 atmosphere. The cells were washed thrice with PBS to remove extracellular fluorescence probes, then centrifuged and suspended in D-PBS. Fluorescence intensity was quantified with a flow cytometer  and FlowJo 7.6.Intracellular ROS levels were determined with 2′,7′-dichlorofluorescin diacetate staining and flow cytometry. Briefly, astrocytes were incubated with 10 μM 2′,7′-dichlorofluorescin diacetate  for 30 min in a humidified 5% CO® Genomic DNA Purification kit  according to the manufacturer’s instructions. Diluted genomic DNA (approximately 20 ng/μL) was digested with formamidopyridine DNA glycosylase (FPG)  in the experimental samples, whereas water was substituted for FPG in the control samples. FPG cleaved oxidized guanine-containing DNA to create a fragmentary template for the subsequent PCR, and Ct values increased. All samples were set up on ice, then incubated at 37 °C for 16 h to allow exhaustive digestion. The 20 μL PCR mixture included 12.5 μL of SYBR Green qPCR Supermix (2×), 1 μL of DNA, 1 μL of telomere forward and reverse primers (10 µM), and PCR-grade water. The reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min [Genomic DNA was extracted using the Wizardor 1 min . PrimersTelomeric DNA oxidative damage was measured using immuno-fluorescence FISH, as described previously . AstrocyTelomere length was determined by southern blot and quantitative real-time PCR (qPCR). Telomere restriction fragment (TRF) analysis was performed using a commercial kit , on the basis of the manufacturer’s instructions with slight modifications. Briefly, DNA (200 ng) was digested with MboI  15 min at 37 °C, then electrophoresed on 1% agarose gel at 6 V for 14 h. The blotting membrane was washed, blocked, incubated with anti-DIG-alkaline phosphatase  for 3 h, washed again, and exposed to a highly sensitive chemiluminescent substrate for 5 min . After exposure of the blot to an X-ray film, terminal restriction fragment analysis was performed with Telo Tool version 1.3.2O (PCR-grade). The reaction mixtures were incubated at 95 °C for 5 min, then subjected to two cycles of 90 °C for 15 s, 49 °C for 15 s, which were followed by 40 amplification cycles . Primers were specific for telomeric gene  and 36B4 . The assay was performed with a Roche 480 sequence detector. The expression of each gene was normalized to that of a reference control gene.Additionally, telomere length was determined by qPCR, as proposed by Cawthon . PCR wasp-value less than 0.05 was considered statistically significant.Data were expressed as mean ± SEM values based on at least three independent experiments. The statistical software package SPSS 24.0 was used to evaluate differences between treatment groups by one-way ANOVA. When significant, ANOVA was followed by a post-hoc test (Tukey’s honestly significant difference test or Dunnet’s test). The correlation coefficients were calculated by spearman correlation analysis. A In conclusion, the novel results presented here are consistent with the hypothesis that folic acid decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. Since astrocyte apoptosis may contribute to the pathogenesis of Alzheimer’s disease and other neurodegenerative disorders ,21, futu


Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fibrosis (IPF), a fatal senescence-associated disease, but to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans.N = 9; 68·7 ± 3·1 years old; 2 female; BMI:33·9 ± 2·3 kg/m2; eGFR:27·0 ± 2·1 mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed.In an open label Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to subjects with diabetic kidney disease (INK4A-and p21CIP1-expressing cells, cells with senescence-associated β-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated by senescent cells, and crown-like structures were decreased. Skin epidermal p16INK4A+ and p21CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and −12.D + Q reduced adipose tissue senescent cell burden within 11 days, with decreases in p16“Hit-and-run” treatment with senolytics, which in the case of D + Q have elimination half-lives <11 h, significantly decreases senescent cell burden in humans.NIH and Foundations.ClinicalTrials.gov Identifier: NCT02848131. Senescence, Frailty, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents. Senescent cells accumulate in tissues with ageing and at etiological sites in multiple chronic diseases, including adipose tissue in diabetes. Senescent cells can release products that cause inflammation and death of non-senescent cells, the senescence-associated secretory phenotype (SASP). Intermittent administration of the senolytic drug combination, Dasatinib plus Quercetin (D + Q), which transiently disables the pro-survival pathways that defend senescent cells against their own apoptotic environment, selectively eliminates senescent cells from mouse and human cell cultures, ageing mice, mice with insulin resistance and many other chronic disorders, and freshly-isolated adipose tissue explants from obese, diabetic human subjects. In the first clinical trial of senolytics, D + Q alleviated physical dysfunction in patients with idiopathic pulmonary fibrosis (IPF), a progressive, fatal, cellular senescence-associated disease. In another clinical trial, prolonged D administration to patients with systemic sclerosis appeared to reduce the SASP and other senescence markers in skin biopsies. To date, no peer-reviewed clinical trial report has directly demonstrated that administering senolytics decreases senescent cells.INK4A-and p21CIP1-expressing cells, cells with senescence-associated β-galactosidase  activity, and adipocyte progenitors with limited replicative potential. Consistent with decreased overall senescent cell burden in humans treated with senolytics, skin epidermal p16INK4A- and p21CIP1-expressing cells were also reduced, as were key SASP factors, including interleukin (IL)-1α, IL-6, and MMPs 9 & 12, in blood. Thus, “hit-and-run” treatment with senolytic agents, which in the case of D + Q have elimination half-lives of <11 h, is sufficient to decrease senescent cell burden in humans. Thereafter, senescent cell burden remains low for days to weeks, consistent with the >2 weeks it takes for new senescent cells to develop (at least in cell culture).Here, in an open-label Phase 1 pilot study, we show for the first time that senolytic drugs decrease senescent cell abundance in humans. A 3-day oral course of D + Q in subjects with diabetic kidney disease (DKD) reduced adipose tissue senescent cell burden 11 days later, as indicated by decreases in cells with markers of senescence: p16Interventions targeting fundamental ageing processes such as cellular senescence could delay, prevent, or alleviate multiple age-related diseases and disorders as a group, instead of one-at-a-time, as per the Geroscience Hypothesis. Increasingly in mice, this hypothesis appears to be true. Combined with the first clinical trial of senolytic agents showing that D + Q improves physical function in patients with IPF published earlier this year in this journal, our current article showing that D + Q actually decreases senescent cell burden in humans is consistent with the possibility that the Geroscience Hypothesis may also hold true for humans. If clinical trials over the next few years support and extend our findings to show that these agents can alleviate additional age- and cellular senescence-related disorders and diseases (beyond IPF) and reduce senescent cell burden (beyond adipose tissue and skin and as reflected by decreased SASP factors in blood), senolytics might become an entirely new path for alleviating currently untreatable chronic diseases and enhancing human healthspan.Alt-text: Unlabelled Box1Cellular senescence is a cell fate that entails essentially irreversible replicative arrest and that was first reported in serially sub-cultured human cells in 1961 . SenesceInk4a-expressing cells, some of which are senescent, can be eliminated by activating the product of the transgene, an approach that is not translatable to humans. Senolytics act by facilitating apoptosis of senescent cells due to their own SASP, not by directly targeting all cells that have high p16Ink4a expression.Senolytics are drugs that specifically target senescent cells by inducing apoptosis of senescent but not non-senescent cells . Work thBy definition, the target of senolytics is senescent cells, not a molecule or a single biochemical pathway. The first senolytic drugs, Dasatinib (D) and Quercetin (Q), were discovered using a mechanism-based approach instead of the random high-throughput screening usually used for drug discovery . ThroughBecause senescent cells can take weeks to months to develop and do not divide, and because even eliminating only 30% of senescent cells can be sufficient to alleviate dysfunction in preclinical studies , D + Q iBased on the many promising studies of D + Q in mice , the expTo test whether intermittent D + Q is effective in targeting senescent cells in humans, we administered a single 3 day course of oral D + Q and assayed senescent cell abundance 11 days after the last dose in subjects with DKD, the most common cause of end-stage kidney failure and which is characterized by increased senescent cell burden . We foun22.12) were included. Individuals with QTc prolongation (>450 msec), liver dysfunction, non-diabetic kidney disease, dialysis, malignancy, active infection, morbid obesity (body mass index [BMI] >50 kg/m2), solid organ transplantation, or taking drugs known to interact with D + Q were excluded. Such drugs include those with a narrow therapeutic range that are substrates for CYP3A4, CYP2C8, CYP2C9, CYP2D6, CYP2C19, CYP1A2, or OATP1B1 or strong inhibitors or inducers of CYP3A4 . Informed consent was obtained from study participants. The Mayo Clinic Institutional Review Board approved this study, which is registered at ClinicalTrials.gov (NCT02848131) and which is an ongoing clinical trial to determine the effectiveness of senolytics for restoring cellular, tissue, and clinical function in patients with diabetic chronic kidney disease. A single 3-day oral treatment regimen with D 100 mg daily and Q 1000 mg total daily (500 mg twice daily) was administered to all participants on Study Days 1 to 3.Study participants were accrued from November 2016 to April 2019 at Mayo Clinic in Rochester, Minnesota. 2.2To avoid sun-exposed areas of skin and to simultaneously obtain abdominal subcutaneous adipose tissue superficial to Scarpa's fascia, elliptical incisional biopsies of adipose tissue and attached skin were acquired from at a point to the right or left of, and 2–5 cm inferior to the navel. Abdominal subcutaneous adipose tissue (0·5–2 g) and attached skin were collected under sterile conditions through a 3–5 cm incision on Study Days 0 and 14. The site for the post-treatment biopsy was contralateral to that for the pre-treatment biopsy, with both the pre- and post-treatment biopsies being from the same dermatome. Skin attached to the adipose tissue biopsy was cut from the underlying adipose tissue. Also on Study Days 0 and 14, blood was collected. Blood samples were stored at −80 °C for subsequent analyses.2.3INK4A , p21CIP1 , CD68 , and CD1a  antibodies using a VENTANA Discovery ULTRA instrument  following instructions and using materials suggested by the manufacturer. Adipose tissue and skin biopsy slides were scanned using a Motic Slide Scanner  and a 40× objective. In adipose tissue biopsies, images scanned for p16INK4A and p21CIP1 were sliced  using Adobe Photoshop  to generate fields that were 400 μm × 300 μm. Thirty fields were selected for analysis from each image using a random number generator. p16INK4A+ and p21CIP1+ cells and adipocytes were counted by ImageJ [+ macrophages, crown-like structures, and adipocyte shells using the software, AMCounter  [INK4A, p21CIP1 , CD1a, and CD68 were counted and divided by the total length of the epidermis (mm) using ImageJ. All image analyses were conducted by observers who were blinded as to whether samples were pre- or post-treatment biopsies.Adipose tissue and skin biopsies were fixed in formalin and paraffin-embedded. Five μm sections were placed on slides and immunohistochemically stained with CINtec Histology monoclonal mouse anti-human p16y ImageJ ,46. For ter, MN) . For ski2.4+ cells were determined as a function of all nuclei in the fields.Adipose biopsies were washed 3 times with PBS, fixed for 5 min in PBS containing 2% (vol/vol) formaldehyde  and 0·25% glutaraldehyde (Sigma-Aldrich), and washed with PBS 3 times. Tissues were incubated in SAβgal activity solution, pH 6·0, at 37 °C for 16–18 h (overnight), washed, stained with Hoechst dye for 10 min for counting nuclei, and kept in PBS until being imaged with a fluorescence microscope . Ten to 15 random fields were imaged per sample. SAβgal+ cells were quantified as above from adipose tissue biopsies collected 15 days apart from 15 obese, premenopausal women who were not taking senolytics and who were enrolled in a separate IRB-approved study .2.6l-glutamine  in a 37 °C/5% CO2 incubator for 3–4 days. When 60–80% confluent, cells were passaged using TrypLE . The third passage cells were collected and kept in Gibco Cell Culture Freezing Medium  at −80 °C. Change in adipocyte progenitor numbers over time was measured in passage 3–4 cultures using a tetrazolium compound  assay  according to the manufacturer's instructions.Adipocyte progenitors, also termed adipose tissue-derived mesenchymal stem cells or preadipocytes, were harvested from abdominal subcutaneous fat samples as previously described ,49. Adip2.7t-test with a 0·05 two-sided significance level. Data are presented as mean ± SE or raw values (tables and text). Welsh's unpaired t-test for unequal variances was used for comparing Day 0 vs. Day 14. The α-level for significance was 0·05. Analyses were conducted using GraphPad Prism 7·02 .Prior to pilot study initiation, sample size estimates were determined. Pilot trial sample size estimation was based on a sample size of 9, which has 80% power to detect a difference in means of 5% , assuming a standard deviation of differences of 4·0, using a 3N = 9; mean age: 69, range 55–79 years; 7 males and 2 females) with drug-controlled diabetes and CKD  adipose tissue p16INK4A+ cells. Raw values were reduced by 35% (p = 0·009) adipose tissue p21CIP1+ cells, with raw values being decreased by 17% (p = 0·005) SAβgal+ cells; raw values were reduced by 62% . D + Q lowered the raw numbers of macrophages per adipocyte by 28%. Senescent cells and macrophages contribute to formation of the “crown-like structures” (CLS) that are characteristic of adipose tissue in diabetes and obesity [Ink4a+ cells [p = 0·001). The raw number of CLS per adipocyte decreased by 86%  to the decreases in p16INK4A+ and SAβgal-expressing cells in adipose tissue after senolytic treatment, we assayed replicative potential of primary adipocyte progenitors isolated from adipose tissue biopsies from the 9 subjects plus an additional 2 subjects . p16INK4A+ cells as a function of epidermal length were 20% less abundant 11 days after completing 3 days of treatment with D + Q than at baseline . Thus, the decrease in epidermal p16INK4A+ cells after D + Q treatment is not readily explained by a decrease in CD68+;p16INK4A+ macrophages or in the related Langerhans cells.To test if D + Q reduces senescent cell burden in tissues in addition to adipose tissue in subjects with diabetes and CKD, we analyzed the epidermal layer of skin overlying the abdominal subcutaneous adipose tissue taken before and after senolytic treatment. Similar to adipose tissue, p16baseline a and p21abundant b. With r p = .8; c. Residep = 0·06). Abundance of adipose tissue p16INK4A+ cells (relative to adipocytes) correlated significantly with plasma IL-6, IL-9, IL-1α, IL1-RA, MMP12, MMP9, and MMP-2 . These circulating SASP factor data are consistent with a systemic decrease in senescent cell abundance that remained apparent well beyond the 4 to 11 h elimination half-lives of D + Q.We tested if key circulating SASP factors were decreased by the short course of senolytics. Plasma IL-1α, −2, −6, and − 9 and Matrix Metalloproteinases (MMP)-2, −9, and −12 were significantly lower 11 days after than before the 3 days of D + Q administration  and IL1-4The intended target of senolytics is senescent cells. We present evidence showing that the drugs, D + Q, which are senolytic in mice and cell or tissue culture models, decrease senescent cells in humans. No individual markers of senescent cells appear to be fully sensitive and specific. Furthermore, individual factors associated with the SASP can be produced by non-senescent cells, such as immune cells. We therefore assayed several key cellular senescence markers and circulating SASP factors in tandem as well as markers of macrophages, which can exhibit features similar to those of senescent cells . MacrophINK4A and p21CIP1 expression  or frailty in elderly women using Fisetin (NCT03325322), an earlier phase trial of D + Q for the accelerated ageing-like state following bone marrow transplantation (NCT02652052), completion of our trial with preliminary results reported here of D + Q for renal insufficiency and stem cell dysfunction in advanced diabetes (NCT02848131), trials for the many other age- and cellular senescence-related disorders using different senolytic agents and combinations that we are about to begin, and trials by others that have yet to be reported following peer review, such as one in which an agent that acts on principally MDM2 is injected into the knees of subjects with osteoarthritis (NCT03513016).Many more clinical trials for different indications and with different senolytic drugs and combinations need to be completed to confirm the findings from the three clinical trials of senolytics reported in peer-reviewed journals so far. These types of clinical studies include later phase trials, such as the randomized, controlled trial we and collaborators are currently conducting using D + Q for IPF , DK118120 (S.M.H.), DK120292 (L.O.L.), AG013925 (J.L.K.), AG062413 , the Translational Geroscience Network (AG061456: J.L.K.), DK45343 (M.D.J.), DK40484 (M.D.J.), and TR002377 , Robert and Arlene Kogod, Satellite Healthcare (L.J.H.), the Connor Group (J.L.K.), Robert J. and Theresa W. Ryan (J.L.K.), and the Glenn (N.K.L.), Ted Nash Long Life (J.L.K.), and Noaber Foundations (J.L.K.). The study sponsors had no role in in the collection, analysis, or interpretation of data; in writing the report; or in the decision to submit the paper for publication. T.T. and J.L.K. confirm they had full access to all the data in the study and had final responsibility for the decision to submit for publication.This work was supported by Study concept, design, and overall supervision: JLK, TT, LJH.Prepared manuscript: JLK, LJH, TT, LGPLP.Edited manuscript: JLK, LJH, EOWG, TT, LGPLP, SK, RJP, MDJ, SKH, SAB, TKE, SMH, DMK, NKL, KMM, SCT, QJ, YZ, JFP.Obtained funding: JLK, LJH.Adipose tissue and skin biopsies: TJM, TAK.Regulatory approvals and oversight: LJH, TKE, KMM, SH, JLK.Patient enrollment, consenting, and clinical assessments: LJH, DKL, SMH, SCT, SAB, EOWG, TLV, JLK.Conducted adipocyte progenitor replication assays and harvesting: KLJ, IMS.Conducted immunohistochemical, SAβgal, and blood laboratory analyses: TT, LGPLP, AX, QJ, MDJ, SGV, ABL, JFP, DMK, KKS.Data analysis: JLK, TT, LJH, LGPLP, RJP.J.L.K., T.T., Y.Z., and N.K.L. have a financial interest related to this research. Patents on senolytic drugs are held by Mayo Clinic. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. No conflicts of interest, financial or otherwise, are declared by the other authors.