五没食子酰基葡萄糖对卵巢癌HO-8910细胞凋亡调控基因表达与胱天蛋白酶凋亡途径的影响

目的探讨五没食子酰基葡萄糖(PGG)诱导卵巢癌HO-8910细胞凋亡的作用及诱导胱天蛋白酶凋亡途径的机制。方法 PGG 10,20,40和80μmol·L-1处理HO-8910细胞48,72和96 h后,MTT法检测细胞存活率;Hoechst 33258染色观察HO-8910细胞核形态改变,AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;Western印迹法检测细胞内胱天蛋白酶酶原及活性形式;RT-PCR检测凋亡调控基因Bax、Bcl-2、Bcl-XL、凋亡抑制因子1(CIAP-1)、CIAP-2、存活蛋白、神经元凋亡抑制蛋白(NIAP)、X连锁凋亡抑制蛋白(XIAP)和细胞周期蛋白D1 mRNA表达。结果 PGG 10～80μmol·L-1分别作用48,72和96 h,随浓度的增加,细胞存活率明显降低,r分别为0.93,0.95和0.86(P<0.05)。PGG 40μmol·L-1使HO-8910细胞的细胞核染色质固缩,出现凋亡形态学改变,早期凋亡率从正常对照组的(0.6±0.1)%分别增加到(3.4±1.1)%,(9.8±3.7)%和(19±4.5)%,对晚期凋亡率影响不明显。PGG 20～80μmol·L-1使HO-8910细胞内胱天蛋白酶3,胱天蛋白酶7和胱天蛋白酶9及其底物多聚腺苷二磷酸核糖聚合酶(PARP)的剪切水平增加,PGG20～80μmol·L-1均抑制死亡受体FAS的蛋白表达水平并使胱天蛋白酶8总剪切水平降低。PGG 20～80μmol·L-1抑制HO-8910细胞中细胞周期蛋白D1,Bcl-2,Bcl-XL和NIAP mRNA的表达,上调CIAP-1 mRNA的表达,对基因Bax,CIAP-2和XIAP mRNA表达影响不明显;PGG 20μmol·L-1抑制存活蛋白基因mRNA的表达,但是增加处理浓度却上调存活蛋白基因mRNA的表达。结论 PGG可能通过抑制凋亡抑制基因Bcl-2和Bcl-XL的表达从而诱导HO-8910细胞内胱天蛋白酶9依赖的内源性凋亡途径,并诱导细胞凋亡。     

姜酮对脂多糖诱导的人脐静脉内皮细胞损伤的影响

摘要：目的:探究姜酮对脂多糖诱导的人脐静脉内皮细胞（HUVECs）损伤的影响。方法:HUVECs经不同浓度的姜酮(0,5,25,50,125,250μmol·L-1)处理24 h后再用LPS(100 ng·ml-1)刺激12 h,之后利用CCK-8试剂盒检测细胞增殖能力并选定合适的姜酮浓度进行后续实验;将HUVECs随机分为4组:对照组;姜酮组(50μmol·L-1);LPS组(100 ngl·ml-1);LPS+姜酮组(100 ng·ml-1+50μmol·L-1)。通过实时荧光定量PCR检测各组细胞的炎症细胞因子[肿瘤坏死因子-α（TNF-α）、白细胞介素1（IL-1）、白细胞介素6（IL-6）]的m RNA表达水平;利用2’,7’-二氯荧光黄双乙酸盐（DCFH-DA）探针对细胞活性氧类（ROS）水平进行检测;采用原位末端缺口标记法（Tunel）对细胞凋亡水平进行检测。结果:不同浓度的姜酮对HUVECs的存活率无明显差异（P>0.05）,而姜酮预处理可逆转LPS刺激导致的存活率下降,其中50,125,250μmol·L-1浓度组差异有统计学意义（P<0.05）。与对照组相比,LPS组的IL-1、IL-6、TNF-αm RNA水平、ROS水平、细胞凋亡数目显著升高（P<0.05）。姜酮预处理后可以抑制LPS诱导的HUVECs炎性反应,改善氧化应激水平,减少细胞凋亡数目（P<0.05）。结论:姜酮对LPS诱导的HUVECs损伤具有保护作用,且其保护作用可能与减少炎性因子表达、抑制氧化应激、减少细胞凋亡数目有关。     

苯扎贝特对内皮细胞LOX-1及抗凋亡基因Bcl-2影响

目的研究苯扎贝特对氧化型低密度脂蛋白(ox-LDL)作用的人脐静脉内皮细胞(HUVECs)血凝素氧化型低密度脂蛋白受体-1(LOX-1)及抗凋亡基因Bcl-2的影响。方法体外培养HUVECs,反转录聚合酶链反应(RT-PCR)观察各组LOX-1及Bcl-2 mRNA的变化,Western-blot方法观察各组Bcl-2蛋白的变化。结果ox-LDL组与正常对照组比较LOX-1表达显著增加(P<0.05),抗凋亡基因Bcl-2表达降低(P<0.05),苯扎贝特组LOX-1 mRNA表达减少(P<0.05),Bcl-2 mRNA和蛋白表达增加(P<0.05)且呈浓度效应依赖关系。结论苯扎贝特可通过下调LOX-1的表达,上调抗凋亡基因Bcl-2表达从而抑制ox-LDL引起的内皮细胞凋亡。     

连翘苷通过调控miR-483-3p对oxLDL诱导的血管平滑肌细胞损伤的影响

摘要：目的:探讨连翘苷(PHN)对氧化型低密度脂蛋白(oxLDL)诱导的血管平滑肌细胞炎症损伤的影响及分子机制。方法:人主动脉血管平滑肌细胞(HA-VSMC)随机分为对照组、oxLDL组(50 mg·ml-1)、oxLDL(50 mg·ml-1)+PHN低(1μmol·L-1)、中(10μmol·L-1)、高(100μmol·L-1)剂量组、oxLDL(50 mg·ml-1)+anti-miR-483-3p组、oxLDL(50 mg·ml-1)+anti-miR-NC组、oxLDL(50 mg·ml-1)+PHN(100μmol·L-1)+miR-483-3p组、oxLDL(50 mg·ml-1)+PHN(100μmol·L-1)+miR-NC组。采用流式细胞术检测细胞凋亡率;Western Blot法检测活化的半胱胺酸蛋白酶(Cleaved-Caspase)3和Cleaved-Caspase9蛋白表达;ELISA法检测白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平;RT-qPCR检测miR-483-3p表达水平。结果:低、中、高浓度连翘苷处理后,oxLDL诱导的HA-VSMC细胞凋亡率、Cleaved-Caspase3和Cleaved-Caspase9蛋白表达水平、IL-1β和TNF-α水平、miR-483-3p表达水平显著降低(P<0.05),且呈浓度依赖性。抑制miR-483-3p降低oxLDL诱导的HA-VSMC细胞凋亡率、Cleaved-Caspase3、Cleaved-Caspase9、IL-1β、TNF-α水平(P<0.05)。过表达miR-483-3p逆转了连翘苷对oxLDL诱导的HA-VSMC炎症损伤的作用。结论:连翘苷通过下调miR-483-3p表达从而抑制oxLDL诱导的HA-VSMC炎症损伤。     

六味地黄方含药血清对H_2O_2致HUVECs损伤的保护作用

目的研究六味地黄方(LWDHF)含药血清对过氧化氢(H2O2)损伤的人脐静脉血管内皮细胞(HUVECs)的保护作用,并阐明其可能的机制。方法采用400μmol·L-1H2O2复制HUVECs损伤模型,LWDHF含药血清分为3个剂量干预组(体积分数为5%、10%、20%);MTT法测定细胞活力,Hoechst染色荧光显微镜观察细胞凋亡形态,Annexin VFITC/PI双染法流式细胞仪检测细胞凋亡情况,RT-PCR法检测Bax、Caspase-3等基因mRNA表达,Western blot法检测Caspase-3、Bax等蛋白表达。结果 LWDHF含药血清能明显促进H2O2损伤后的HUVECs增殖;Hoechst染色和Annexin V-FITC/PI双染法结果均显示LWDHF含药血清抑制血管内皮细胞凋亡;RT-PCR和Western blot结果提示LWDHF抑制H2O2所致的血管内皮细胞凋亡可能是通过降低Caspase-3、Bax mRNA和蛋白的表达,升高Bcl-2 mRNA和蛋白表达实现的。结论 LWDHF含药血清可以抑制H2O2诱导的细胞凋亡,对血管内皮细胞具有一定的保护作用。     

非诺贝特对血管紧张素II诱导RAW264.7细胞Toll样受体4表达, 髓过氧化物酶活性及表达的抑制作用

探讨非诺贝特对血管紧张素Ⅱ (AngⅡ) 诱导的小鼠巨噬细胞株RAW264.7细胞Toll样受体4 (TLR4) mRNA、蛋白表达及髓过氧化物酶 (MPO) 活性、mRNA及蛋白表达的影响及其抗炎机制。采用RT-PCR检测TLR4和MPO mRNA水平, Western blotting检测TLR4和MPO的蛋白表达,比色法测定细胞培养上清液中的MPO活性。结果显示,非诺贝特浓度依赖性地减少AngⅡ诱导的RAW264.7细胞TLR4 mRNA及蛋白表达,抑制AngⅡ诱导的RAW264.7细胞MPO活性、mRNA及蛋白表达。此外,TLR4阻断剂对AngⅡ诱导的RAW264.7细胞MPO活性有部分的抑制作用,而非诺贝特可增强这一抑制效应。同时非诺贝特明显拮抗TLR4特异性配体脂多糖的促MPO分泌效应。以上结果表明,非诺贝特可下调AngⅡ诱导的RAW264. 7细胞TLR4表达,并通过干预TLR4,影响胞内信号转导途径,抑制MPO分泌,减轻炎症反应,这可能是其新的抗炎机制之一。


     

川东獐牙菜素A保护溶血性磷脂酰胆碱诱导的内皮细胞损伤

目的 观察川东獐牙菜素A对溶血性磷脂酰胆碱(LPC)诱导的血管内皮细胞损伤的保护。方法 ①检测Cu2+诱导的低密度脂蛋白(LDL)氧化和二苯代苦味肼基自由基(DPPH)反应。②在大鼠离体胸主动脉环,检测乙酰胆碱诱导的内皮依赖性舒张反应。③培养内皮细胞,测定培养液中乳酸脱氢酶(LDH)、丙二醛(MDA)、一氧化氮(NO)和非对称性二甲基精氨酸(ADMA)浓度及细胞内二甲精氨酸二甲胺水解酶(DDAH)活性。结果①川东獐牙菜素A能显著抑制Cu2+诱导的LDL氧化和清除DPPH。②川东獐牙菜素A(10或30μmol·L-1)能改善LPC(5 mg·L-1)所致血管内皮依赖性舒张功能损伤。⑧川东獐牙菜素A(1、3或10μmol·L-1)能抑制LPC(5 mg·L-1)诱导的培养液中LDH和MDA浓度的降低以及NO浓度的增加;川东獐牙菜素A(3或10 μmol·L-1)能显著抑制LPC诱导的ADMA水平升高;川东獐牙菜素A(10μmol·L-1)能显著增加DDAH活性。结论 川东獐牙菜素A对LPC所致的血管内皮细胞损伤有保护作用,其作用与增加DDAH活性和降低AD-MA浓度有关。     

MG132诱导人血管内皮细胞凋亡及对caspase-3表达的影响

目的　观察蛋白酶体抑制剂MG132对人血管脐静脉内皮细胞(ECV -304)的致凋亡作用及其对凋亡相关的天冬氨酸特异的半胱氨酸蛋白酶3表达的影响。方法　采用两个浓度(2, 5μmol·L-1 )的蛋白酶体抑制剂MG132处理ECV -304细胞;DNA琼脂糖凝胶电泳检测细胞凋亡,流式细胞术检测细胞周期和细胞凋亡率;RT -PCR检测细胞内凋亡相关基因caspase -3的转录水平;免疫细胞化学检测细胞caspase 3蛋白表达。结果　对照组ECV -304细胞凋亡率低于5%,在2μmol·L-1MG132作用下,凋亡率为11. 3%,MG132浓度升至5μmol·L-1,细胞凋亡率增致44 .5%,MG132诱导ECV 304细胞凋亡具有量-效关系;RT -PCR检测发现细胞内凋亡相关基因caspase 3mRNA表达上调;免疫细胞化学检测细胞caspase- 3蛋白表达水平升高。结论:蛋白酶体抑制剂MG132能够诱导血管内皮细胞凋亡,其机制可能与MG132抑制UPP活性,促进caspase- 3基因转录,使细胞内caspase -3增加而促进细胞凋亡。     

普伐他汀对溶血磷脂酰胆碱促血管平滑肌细胞增殖及细胞粘附的影响

目的　探讨羟甲基戊二酰辅酶A还原酶抑制剂普伐他汀对溶血磷脂酰胆碱 (LPC)促血管平滑肌细胞增殖及白细胞与内皮细胞粘附的影响。方法　用MTT法检测LPC对平滑肌细胞增殖的量效和时效关系及普伐他汀对LPC促平滑肌细胞增殖的影响 ;用直接记数法检测LPC诱导中性粒细胞系K5 6 2细胞与人脐静脉内皮细胞系ECV30 4细胞的粘附和普伐他汀对LPC所致白细胞与内皮细胞粘附的影响。结果　用 1～ 10 μmol·L-1LPC孵育血管平滑肌细胞 2 4～ 4 8h后 ,呈时间和剂量依赖性地诱导细胞增殖 ,而用 0 3～ 1mmol·L-1普伐他汀预孵育平滑肌细胞 1h ,再与 3μmol·L-1LPC共孵育 2 4～ 4 8h ,明显地抑制LPC所诱导的细胞增殖 ;用 3μmol·L-1LPC孵育ECV30 4细胞 12h显著增加K5 6 2细胞与ECV30 4细胞的粘附 ,而将ECV30 4细胞用 1mmol·L-1普伐他汀预处理后 ,明显减少白细胞与内皮细胞的粘附。结论　普伐他汀可抑制LPC促血管平滑肌细胞增殖及白细胞与内皮细胞粘附的作用。     

苯扎贝特通过过氧化物酶体增殖物激活受体α信号途径对人横纹肌RD细胞的毒性作用

目的探讨苯扎贝特对人骨骼肌细胞的毒性作用及其作用机制。方法人横纹肌RD细胞与苯扎贝特0(对照组),10,30,100,300和1000μmol·L-1作用24h,WST-1法检测细胞存活;苯扎贝特与MK8861μmol·L-1共同作用RD细胞24h,实时荧光定量PCR检测过氧化物酶体增殖物激活受体α(PPARα)与丙酮酸脱氢酶激酶4(PDK4)mRNA的表达。结果苯扎贝特10～100μmol·L-1对RD细胞存活率无影响,300和1000μmol·L-1明显抑制RD细胞的生长,抑制率分别为13%和31%(P<0.01)。苯扎贝特300和1000μmol·L-1可明显升高PPARαmRNA表达,分别为对照组的2和3倍(P<0.05);苯扎贝特联合MK886组,PPARαmRNA表达与正常对照组无差异,但可显著抑制苯扎贝特300和1000μmol·L-1的升高作用(P<0.05)。与正常对照组相比,苯扎贝特单独或与MK886联合组,PDK4mRNA的表达均显著增加(P<0.01);与苯扎贝特单独组比较,苯扎贝特30,100,300和1000μmol·L-1与MK886合用组PDK4mRNA表达分别下降了44%,60%,69%和63%(P<0.05)。结论苯扎贝特对RD细胞具有明显的毒性作用,其作用机制可能是PPARα发挥了重要的调节作用。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

2-(Acetoxyphenyl)-(Z)-styryl sulfides as cyclooxygenase inhibitors

2-(Acetoxyphenyl)-(Z)-styryl sulfides are described as selective cyclooxygenase-2 (COX-2) inhibitors, useful for treating inflammation and COX-2-mediated disorders including neoplasia. 2-(Acetoxyphenyl)-(Z)-styryl sulfide is claimed to be the most potent COX inhibitor in the series with a COX-2 selectivity ratio of 33. This compound is also claimed to be superior to celecoxib (Celebrex^®, Pfizer) in inhibiting cell growth of colorectal carcinoma cells. In this evaluation, the COX inhibitory activity of this compound is compared to that previously disclosed for diarylheterocycles and 2-(acetoxyphenyl)alkyl sulfides. The validity of the DLD-1 cell line in the growth inhibition studies is questioned based on recent literature reports indicating the lack of COX-2 expression in this cell line.     

Sleep-disordered breathing with chronic opioid use

Chronic opioid use for pain relief or as substitution therapy for illicit drug abuse is prevalent in our societies. In the US, retail distribution of methadone and oxycodone has increased by 824 and 660%, respectively, between 1997 and 2003. μ-Opioids depress respiration and deaths related to illicit and non illicit chronic opioid use are not uncommon. Since 2001 there has been an emerging literature that suggests that chronic opioid use is related to central sleep apnoea of both periodic and non-periodic breathing types, and occurs in ～ 30% of these subjects. The clinical significance of these sleep-related abnormalities are unknown. This review addresses the present knowledge of control of ventilation mechanisms during wakefulness and sleep, the effects of opioids on ventilatory control mechanisms, the sleep-disordered breathing found with chronic opioid use and a discussion regarding the future research directions in this area.     

Drug targets for cognitive enhancement in neuropsychiatric disorders

The investigation of novel drug targets for treating cognitive impairments associated with neurological and psychiatric disorders remains a primary focus of study in central nervous system (CNS) research. Many promising new therapies are progressing through preclinical and clinical development, and offer the potential of improved treatment options for neurodegenerative diseases such as Alzheimer's disease (AD) as well as other disorders that have not been particularly well treated to date like the cognitive impairments associated with schizophrenia (CIAS). Among targets under investigation, cholinergic receptors have received much attention with several nicotinic agonists (α7 and α4β2) actively in clinical trials for the treatment of AD, CIAS and attention deficit hyperactivity disorder (ADHD). Both glutamatergic and serotonergic (5-HT) agonists and antagonists have profound effects on neurotransmission and improve cognitive function in preclinical experiments with animals; some of these compounds are now in proof-of-concept studies in humans. Several histamine H3 receptor antagonists are in clinical development not only for cognitive enhancement, but also for the treatment of narcolepsy and cognitive deficits due to sleep deprivation because of their expression in brain sleep centers. Compounds that dampen inhibitory tone (e.g., GABA_A α5 inverse agonists) or elevate excitatory tone (e.g., glycine transporter inhibitors) offer novel approaches for treating diseases such as schizophrenia, AD and Down syndrome. In addition to cell surface receptors, intracellular drug targets such as the phosphodiesterases (PDEs) are known to impact signaling pathways that affect long-term memory formation and working memory. Overall, there is a genuine need to treat cognitive deficits associated with many neuropsychiatric conditions as well as an increasingly aging population.