The Role of in silico Research in Developing Nanoparticle-Based Therapeutics.

Nanoparticles (NPs) hold great potential as therapeutics, particularly in the realm of drug delivery. They are effective at functional cargo delivery and offer a great degree of amenability that can be used to offset toxic side effects or to target drugs to specific regions in the body. However, there are many challenges associated with the development of NP-based drug formulations that hamper their successful clinical translation. Arguably, the most significant barrier in the way of efficacious NP-based drug delivery systems is the tedious and time-consuming nature of NP formulation-a process that needs to account for downstream effects, such as the onset of potential toxicity or immunogenicity, in vivo biodistribution and overall pharmacokinetic profiles, all while maintaining desirable therapeutic outcomes. Computational and AI-based approaches have shown promise in alleviating some of these restrictions. Via predictive modeling and deep learning, in silico approaches have shown the ability to accurately model NP-membrane interactions and cellular uptake based on minimal data, such as the physicochemical characteristics of a given NP. More importantly, machine learning allows computational models to predict how specific changes could be made to the physicochemical characteristics of a NP to improve functional aspects, such as drug retention or endocytosis. On a larger scale, they are also able to predict the in vivo pharmacokinetics of NP-encapsulated drugs, predicting aspects such as circulatory half-life, toxicity, and biodistribution. However, the convergence of nanomedicine and computational approaches is still in its infancy and limited in its applicability. The interactions between NPs, the encapsulated drug and the body form an intricate network of interactions that cannot be modeled with absolute certainty. Despite this, rapid advancements in the area promise to deliver increasingly powerful tools capable of accelerating the development of advanced nanoscale therapeutics. Here, we describe computational approaches that have been utilized in the field of nanomedicine, focusing on approaches for NP design and engineering.

Decline in Helicoverpa armigera nucleopolyhedrovirus occlusion body yields with increasing infection cell density in vitro is strongly correlated with viral DNA levels.

The phenomenon of the reduction in the cell-specific yield with increasing infection cell density (ICD), the cell density effect, is one of the main hurdles for improving virus yields in vitro. In the current study, the reduction in cell-specific yields (viral DNA [vDNA], polyhedrin mRNA and occlusion body [OB]) with increasing ICD for Helicoverpa armigera nucleopolyhedrovirus (HearNPV)-infected HzAM1 (Helicoverpa zea) insect cells has been investigated. HzAM1 cells were propagated in Sf900™ III serum-free medium and synchronously infected with wild-type HearNPV at various ICDs of 0.5-5 × 10(6) cells/mL at an MOI of 5 PFU/cell. Infection was conducted either in the original medium or in fresh medium. As found previously for Sf9 and High Five cells, there were negative correlations between the three key virus infection indicators (vDNA, mRNA and OB) and the peak cell density (PCD). Generally, the yield decline with increasing PCD was most pronounced for OB, followed by mRNA, and was more moderate for vDNA. The decline was significantly reduced, but not totally arrested, when fresh medium was used. There were also strong correlations between OB and mRNA, mRNA and vDNA, and OB and vDNA levels. These results suggest that the reduction in baculovirus yield (OB) at high PCDs is associated with limitations during the upstream processes of replication and transcription together with limitations during protein translation. Furthermore, the peak protein productivity per unit of cell volume in the HzAM1/HearNPV system was shown to be higher than that of the Sf9/rAcMNPV system, but lower than that of the High Five/rAcMNPV system.

Chronic Stress Decreases Basal Levels of Memory-Related Signaling Molecules in Area CA1 of At-Risk (Subclinical) Model of Alzheimer's Disease.

An important factor that may affect the severity and time of onset of Alzheimer's disease (AD) is chronic stress. Epidemiological studies report that chronically stressed individuals are at an increased risk for developing AD. The purpose of this study was to reveal whether chronic psychosocial stress could hasten the appearance of AD symptoms including changes in basal levels of cognition-related signaling molecules in subjects who are at risk for the disease. We investigated the effect of chronic psychosocial stress on basal levels of memory-related signaling molecules in area CA1 of subclinical rat model of AD. The subclinical symptomless rat model of AD was induced by osmotic pump continuous intracerebroventricular (ICV) infusion of 160 pmol/day Aß1-42 for 14 days. Rats were chronically stressed using the psychosocial stress intruder model. Western blot analysis of basal protein levels of important signaling molecules in hippocampal area CA1 showed no significant difference between the subclinical AD rat model and control rat. Following six weeks of psychosocial stress, molecular analysis showed that subclinical animals subjected to stress have significantly reduced basal levels of p-CaMKII and decreased p-CaMKII/t-CaMKII ratio as well as decreased basal levels of p-CREB, total CREB, and BDNF. The present results suggest that these changes in basal levels of signaling molecules may be responsible for impaired learning, memory, and LTP in this rat model, which support the proposition that chronic stress may accelerate the emergence of AD in susceptible individuals.

Effect of the peak cell density of recombinant AcMNPV-infected Hi5 cells on baculovirus yields.

The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and ß-galactosidase (ß-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the ß-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.5 and 6 × 10(6) cells/mL and a multiplicity of infection of 10 plaque-forming units (PFU)/cell either in the original or fresh medium conditions. There were negative correlations between the three key virus infection indicators (vDNA, mRNA and ß-Gal) and the peak cell density (PCD). However, unlike infected Sf9 cells, the yield decline started at the lowest PCD investigated (0.6 × 10(6) cells/mL). Generally, the yield decline with increasing PCD was most pronounced for ß-Gal followed by mRNA and was more moderate for vDNA. The decline was significantly reduced but not totally arrested when fresh medium replacement was used. The results suggest that the reduction in recombinant protein-specific yields at high PCDs is associated with limitations during the up-stream processes of replication and transcription rather than entirely caused by limitations during translation. In addition, low production rates at late infection stages of moderate to high ICDs are a probable cause of the cell density effect.

Chronic psychosocial stress impairs early LTP but not late LTP in the dentate gyrus of at-risk rat model of Alzheimer's disease.

The CA1 and dentate gyrus regions of the hippocampus are physically and functionally closely related but they react differently to insults. This study examined the effect of chronic psychosocial stress on the dentate gyrus of an at-risk (preclinical) rat model of Alzheimer's disease (subAß rats). Chronic psychosocial stress was produced using a rat intruder model. The at-risk rat model of Alzheimer's disease was created by osmotic pump infusion of sub-pathological dose of Aß (160 pmol Aß1-42/day i.c.v) for 14 days. Electrophysiological methods were used to evoke and record early and late phase LTP in the dentate gyrus of anesthetized rats, and immunoblotting was used to measure levels of memory-related signaling molecules in the same region. Electrophysiological and molecular tests in the dentate gyrus showed that subAß rats or stressed rats were not different from control rats. However, when the subAß rats were chronically stressed, the combined treatments severely suppressed early phase LTP without affecting the late phase LTP of dentate gyrus. Additionally, in the chronically stressed subAß rats the expected elevation of levels of phosphorylated CaMKII did not materialize after expression of early phase LTP suggesting impaired phosphorylation, which may explain the severely blocked early phase LTP.

Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression.

The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant LacZ mRNA, and ß-galactosidase (ß-gal) protein, over a wide range of cell densities. Sf9 suspension cell cultures were propagated in Sf-900 III serum-free medium and synchronously infected with rAcMNPV at multiple infection cell densities (ICDs) of between 0.5 and 8 × 10(6) cells/mL. There was a strong negative linear correlation between the specific ß-gal yield and the peak cell density (PCD) post-infection, but contrary to previous reports, the yield decline started at a lower PCD of around 1 × 10(6) cells/mL. Most interestingly, there also was a corresponding strong negative linear correlation between the specific vDNA or LacZ mRNA yield, and the PCD. Comparing the infections at the highest and lowest PCDs tested, the yield decline was most dramatic for ß-gal protein (95 %) and LacZ mRNA (90 %), while it was more moderate for vDNA (50 %). These declines were significantly reduced but not completely arrested, when spent medium was replaced with fresh at the ICD. These findings suggest that protein yield deterioration with increasing cell density originated from limitations during upstream events such as virus gene replication or transcription, rather than during the translational phase. Such limitations may be largely nutritional, but a more complex mechanism may be implicated.

Role of α7- and α4ß2-nAChRs in the neuroprotective effect of nicotine in stress-induced impairment of hippocampus-dependent memory.

We have previously shown that nicotine prevents stress-induced memory impairment. In this study, we have investigated the role of α7- and α4ß2-nicotinic acetylcholine receptors (nAChRs) in the protective effect of nicotine during chronic stress conditions. Chronic psychosocial stress was induced using a form of rat intruder model. During stress, specific antagonist for either α7-nAChRs [methyllycaconitine (MLA)] or α4ß2-nAChRs [dihydro-ß-erythroidine (DHßE)] was infused into the hippocampus using a 4-wk osmotic pump at a rate of 82 µg/side.d and 41 µg/side.d, respectively. Three weeks after the start of infusion, all rats were subjected to a series of cognitive tests in the radial arm water maze (RAWM) for six consecutive days or until the animal reached days to criterion (DTC) in the fourth acquisition trial and in all memory tests. DTC is defined as the number of days the animal takes to make no more than one error in three consecutive days. In the short-term memory test, MLA-infused stressed/nicotine-treated rats made similar errors to those of stress and significantly more errors compared to those of stress/nicotine, nicotine or control groups. This finding was supported by the DTC values for the short memory tests. Thus, MLA treatment blocked the neuroprotective effect of nicotine during chronic stress. In contrast, DHßE infusion did not affect the RAWM performance of stress/nicotine animals. These results strongly suggest the involvement of α7-nAChRs, but not α4ß2-nAChRs, in the neuroprotective effect of chronic nicotine treatment during chronic stress conditions.

Improving the robustness of a low-cost insect cell medium for baculovirus biopesticides production, via hydrolysate streamlining using a tube bioreactor-based statistical optimization routine.

A critical component of an in vitro production process for baculovirus biopesticides is a growth medium that is efficacious, robust, and inexpensive. An in-house low-cost serum-free medium, VPM3, has been shown to be very promising in supporting Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) production in H. zea insect cell suspension cultures, for use as a biopesticide against the Heliothine pest complex. However, VPM3 is composed of a significant number of undefined components, including five different protein hydrolysates, which introduce a challenging lot-to-lot variability to the production process. In this study, an intensive statistical optimization routine was employed to reduce the number of protein hydrolysates in VPM3 medium. Nearly 300 runs (including replicates) were conducted with great efficiency by using 50 mL TubeSpin® bioreactors to propagate insect cell suspension cultures. Fractional factorial experiments were first used to determine the most important of the five default protein hydrolysates, and to screen for seven potential substitutes for the default meat peptone, Primatone RL. Validation studies informed by the screening tests showed that promising alternative media could be formulated based on just two protein hydrolysates, in particular the YST-AMP (Yeast Extract and Amyl Meat Peptone) and YST-POT (Yeast Extract and Lucratone Potato Peptone) combinations. The YST-AMP (meat-based) and YST-POT (meat-free) variants of VPM3 were optimized using response surface methodology, and were shown to be just as good as the default VPM3 and the commercial Sf-900 II media in supporting baculovirus yields, hence providing a means toward a more reproducible and scalable production process for HaSNPV biopesticides.

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells.

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus-insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell-baculovirus systems for bioprocess optimization.

Elevation of BACE in an Aß rat model of Alzheimer's disease: exacerbation by chronic stress and prevention by nicotine.

In Alzheimer's disease (AD), progressive accumulation of ß-amyloid (Aß) peptides impairs nicotinic acetylcholine receptor (nAChR) function by a mechanism that may involve α7 and α4ß2-nAChR subtypes. Additionally, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE), the rate-limiting enzyme in the pathogenic Aß production pathway, is expressed at high levels in hippocampal and cortical regions of AD brains. We measured hippocampal area CA1 protein levels of BACE and α7- and α4ß2-nAChR subunits using an Aß rat model of AD (14-d osmotic pump i.c.v. infusion of 300 pmol/d Aß peptides) in the presence and absence of chronic stress and/or chronic nicotine treatment. There was a significant increase in the levels of BACE in Aß-infused rats, which were markedly intensified by chronic (4-6 wk) stress, but were normalized in Aß rats chronically treated with nicotine (1 mg/kg b.i.d.). The levels of the three subunits α7, α4 and ß2 were significantly decreased in Aß rats, but these were also normalized in Aß rats chronically treated with nicotine. Chronic stress did not further aggravate the reduction of nAChRs in Aß-infused rats. The increased BACE levels and decreased nAChR levels, which are established hallmarks of AD, provide additional support for the validity of the Aß i.c.v.-infused rat as a model of AD.

Chronic psychosocial stress enhances long-term depression in a subthreshold amyloid-beta rat model of Alzheimer's disease.

In addition to genetic aspects, environmental factors such as stress may also play a critical role in the etiology of the late onset, sporadic Alzheimer's disease (AD). The present study examined the effect of chronic psychosocial stress in a sub-threshold Aß (subAß) rat model of AD on long-term depression by two techniques: electrophysiological recordings of synaptic plasticity in anesthetized rats, and immunoblot analysis of memory- and AD-related signaling molecules. Chronic psychosocial stress was induced using a rat intruder model. The subAß rat model of AD, which was intended to represent outwardly normal individuals with a pre-disposition to AD, was induced by continuous infusion of 160 pmol/day Aß1₋42 via a 14-day i.c.v. osmotic pump. Results from electrophysiological recordings showed that long-term depression evoked in stress/subAß animals was significantly enhanced compared with that in animals exposed to stress or subAß infusion alone. Molecular analysis of various signaling molecules 1 h after induction of long-term depression revealed an increase in the levels of calcineurin and phosphorylated CaMKII in groups exposed to stress compared with other groups. The levels of the brain-derived neurotrophic factor (BDNF) were significantly decreased in stress/subAß animals but not in stress or subAß animals. In addition, the levels of beta-site amyloid precursor protein cleaving enzyme were markedly increased in stress/subAß. These findings suggest that chronic stress may accelerate the impairment of synaptic plasticity and consequently cognition in individuals 'at-risk' for AD.

Chronic psychosocial stress exacerbates impairment of synaptic plasticity in ß-amyloid rat model of Alzheimer's disease: prevention by nicotine.

Alzheimer's disease (AD) is a degenerative disorder that leads to progressive, irreversible cognitive decline. It develops as a result of over-production and aggregation of ß-amyloid (Aß) peptides in the brain. We have recently shown that stress exacerbates, while nicotine prevents long-term memory impairment induced by ß-Amyloid. In this study, we evaluated the effect of chronic psychosocial stress on synaptic plasticity (Late-phase long-term potentiation; L-LTP, and long-term depression; LTD) in the ß-Amyloid rat model of AD, and the positive impact of chronic nicotine treatment. Chronic psychosocial stress was induced by an intruder method. The Rat AD model was induced by 14-day i.c.v. osmotic pump infusion of a 1:1 mixture of 300 pmol/day Aß1-40/Aß1-42. The rats were treated with nicotine (2 mg/kg/day) for 6 weeks. In vivo electrophysiological recordings of L-LTP, and LTD in hippocampal area CA1 showed that chronic stress by itself did not affect L-LTP. However, it markedly aggravated the impairment of this response as well as LTD in Aß- treated rats. The effects of Aß and the combination of stress and Aß were totally prevented by chronic nicotine treatment. Immunoblot analysis revealed that stress and/or Aß significantly increased the basal levels of calcineurin and prevented the expected L-LTP-induced increase in CREB phosphorylation, and CaMKIV levels. These effects were not seen in Aß- infused rats chronically treated with nicotine. The changes in synaptic plasticity-related molecules may explain the effects of stress and/or chronic nicotine on L-LTP in Aß animals.

Sleep deprivation prevents stimulation-induced increases of levels of P-CREB and BDNF: protection by caffeine.

It is well known that caffeine and sleep deprivation have opposing effects on learning and memory; therefore, this study was undertaken to determine the effects of chronic (4wks) caffeine treatment (0.3g/l in drinking water) on long-term memory deficit associated with 24h sleep deprivation. Animals were sleep deprived using the modified multiple platform method. The results showed that chronic caffeine treatment prevented the impairment of long-term memory as measured by performance in the radial arm water maze task and normalized L-LTP in area CA1 of the hippocampi of sleep-deprived anesthetized rats. Sleep deprivation prevents the high frequency stimulation-induced increases in the levels of phosphorylated-cAMP response element binding protein (P-CREB) and brain-derived neurotrophic factor (BDNF) seen during the expression of late phase long-term potentiation (L-LTP). However, chronic caffeine treatment prevented the effect of sleep-deprivation on the stimulated levels of P-CREB and BDNF. The results suggest that chronic caffeine treatment may protect the sleep-deprived brain probably by preserving the levels of P-CREB and BDNF.

Chronic psychosocial stress accelerates impairment of long-term memory and late-phase long-term potentiation in an at-risk model of Alzheimer's disease.

Although it is generally agreed that Aß contributes to the pathogenesis of AD, its precise role in AD and the reason for the varying intensity and time of onset of the disease have not been elucidated. In addition to genetic factors, environmental issues such as stress may also play a critical role in the etiology of AD. This study examined the effect of chronic psychosocial stress in an at-risk (treatment with a subpathogenic dose of Aß; "subAß") rat model of AD on long-term memory by three techniques: memory tests in the radial arm water maze, electrophysiological recordings of synaptic plasticity in anesthetized rats, and immunoblot analysis of learning- and long-term memory-related signaling molecules. Chronic psychosocial stress was induced using a rat intruder model. The subAß rat model of AD was induced by continuous infusion of 160 pmol/day Aß(1-42) via a 14-day i.c.v. osmotic pump. All tests showed that subAß rats were not different from control rats. Result from behavioral tests and electrophysiological recordings showed that infusion of subAß in chronically stressed rats (stress/subAß group) caused significant impairment of cognitive functions and late-phase long-term potentiation (L-LTP). Molecular analysis of various signaling molecules after expression of L-LTP, revealed an increase in the levels of p-CREB in control, stress, and subAß rats, but not in the stress/subAß rats. These findings suggest that the chronic stress-induced molecular alteration may accelerate the impairment of cognition and synaptic plasticity in individuals "at-risk" for AD.

Chronic nicotine restores normal Aß levels and prevents short-term memory and E-LTP impairment in Aß rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by increased deposition of beta-amyloid (Aß) peptides and progressive cholinergic dysfunction in regions of the brain involved in learning and memory processing. In AD, progressive accumulation of Aß peptide impairs nicotinic acetylcholine receptor (nAChR) function by an unknown mechanism believed to involve α(7)- and α(4)ß(2)-nAChR blockade. The three approaches of the current study evaluated the effects of chronic nicotine treatment in the prevention of Aß-induced impairment of learning and short-term memory. Rat AD model was induced by 14-day i.c.v. osmotic pump infusion of a 1:1 mixture of 300 pmol/day Aß(1-40)/Aß(1-42) or Aß(40-1) (inactive peptide, control). The effect of nicotine (2 mg/(kg day)) on Aß-induced spatial learning and memory impairments was assessed by evaluation of performance in the radial arm water maze (RAWM), in vivo electrophysiological recordings of early-phase long-term potentiation (E-LTP) in urethane-anesthetized rats, and immunoblot analysis to determine changes in the levels of beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE), Aß and memory-related proteins. The results indicate that 6 weeks of nicotine treatment reduced the levels of Aß(1-40) and BACE1 peptides in hippocampal area CA1 and prevented Aß-induced impairment of learning and short-term memory. Chronic nicotine also prevented the Aß-induced inhibition of basal synaptic transmission and LTP in hippocampal area CA1. Furthermore, chronic nicotine treatment prevented the Aß-induced reduction of α(7)- and α(4)-nAChR. These effects of nicotine may be due, at least in part, to upregulation of brain derived neurotropic factor (BDNF).

Intensification of long-term memory deficit by chronic stress and prevention by nicotine in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cholinergic dysfunction and deposition of beta-amyloid (Aß) in regions of the brain associated with learning and memory. The sporadic nature and late onset of most AD cases suggests that aside from biological determinants, environmental factors such as stress may also play a role in the progression of the disease. Behavioral and molecular studies were utilized to evaluate the effects of chronic nicotine treatment in the prevention of impairment of long-term memory. The rat model of AD was induced by i.c.v. osmotic pump infusion of Aß peptides. Chronic psychosocial stress and chronic nicotine treatment were instituted for 6weeks. Spatial memory testing in the Radial Arm Water Maze revealed that, although stress, by itself, did not affect long-term memory, the combination of chronic stress and Aß infusion impaired long-term memory significantly more than Aß peptides infusion alone. Chronic nicotine treatment completely prevented Aß- and stress/Aß combination-induced memory impairment. Furthermore, molecular findings in hippocampal CA1 region of stress/Aß rats indicated marked reduction in the protein levels of phosphorylated cAMP response element binding (p-CREB) and calcium-calmodulin-dependent protein kinase IV (CaMKIV), with significant increases in the levels of brain-derived neurotrophic factor (BDNF). These disturbances in signaling pathways, which may be the underlying mechanisms of impairment of long-term memory in these rats, were totally prevented by chronic nicotine treatment.

Caffeine prevents sleep loss-induced deficits in long-term potentiation and related signaling molecules in the dentate gyrus.

We have previously reported that caffeine prevented sleep deprivation-induced impairment of long-term potentiation (LTP) of area CA1 as well as hippocampus-dependent learning and memory performance in the radial arm water maze. In this report we examined the impact of long-term (4-week) caffeine consumption (0.3 g/L in drinking water) on synaptic plasticity (Alhaider et al., 2010) deficit in the dentate gyrus (DG) area of acutely sleep-deprived rats. The sleep deprivation and caffeine/sleep deprivation groups were sleep-deprived for 24 h by using the columns-in-water technique. We tested the effect of caffeine and/or sleep deprivation on LTP and measured the basal levels as well as stimulated levels of LTP-related molecules in the DG. The results showed that chronic caffeine administration prevented the impairment of early-phase LTP (E-LTP) in the DG of sleep-deprived rats. Additionally, chronic caffeine treatment prevented the sleep deprivation-associated decreases in the basal levels of the phosphorylated calcium/calmodulin-dependent protein kinase II (P-CaMKII) and brain derived neurotrophic factor (BDNF) as well as in the stimulated levels of P-CaMKII in the DG area. The results suggest that chronic use of caffeine prevented anomalous changes in the basal levels of P-CaMKII and BDNF associated with sleep deprivation and as a result contributes to the revival of LTP in the DG region.

Chronic caffeine treatment prevents sleep deprivation-induced impairment of cognitive function and synaptic plasticity.

STUDY OBJECTIVES: This study was undertaken to provide a detailed account of the effect of chronic treatment with a small dose of caffeine on the deleterious effects of sleep loss on brain function in rats. EXPERIMENTAL DESIGN: We investigated the effects of chronic (4 weeks) caffeine treatment (0.3 g/L in drinking water) on memory impairment in acutely (24 h) sleep-deprived adult male Wistar rats. Sleep deprivation was induced using the modified multiple platform model. The effects of caffeine on sleep deprivation-induced hippocampus-dependent learning and memory deficits were studied by 3 approaches: learning and memory performance in the radial arm water maze task, electrophysiological recording of early long-term potentiation (E-LTP) in area CA1 of the hippocampus, and levels of memory- and synaptic plasticity-related signaling molecules after E-LTP induction. MEASUREMENT AND RESULTS: The results showed that chronic caffeine treatment prevented impairment of hippocampus-dependent learning, shortterm memory and E-LTP of area CA1 in the sleep-deprived rats. In correlation, chronic caffeine treatment prevented sleep deprivation-associated decrease in the levels of phosphorylated calcium/calmodulin-dependent protein kinase II (P-CaMKII) during expression of E-LTP. CONCLUSIONS: The results suggest that long-term use of a low dose of caffeine prevents impairment of short-term memory and E-LTP in acutely sleep-deprived rats.

Chronic psychosocial stress triggers cognitive impairment in a novel at-risk model of Alzheimer's disease.

Although it is generally accepted that Abeta contributes to the pathogenesis of Alzheimer's disease (AD), other factors that impact the severity and time of onset of the disease are not well known. Aside from genetic factors, environmental factors such as stress may also play a critical role in the manifestation of AD. The present study examined the effect of chronic psychosocial stress in an at-risk, subthreshold Abeta (subAbeta) rat model of AD by three approaches: learning and memory tests in the radial arm water maze, electrophysiological recordings of long-term potentiation (LTP) in anesthetized rats, and immunoblot analysis of learning- and memory-related signaling molecules. Chronic psychosocial stress was induced using a rat intruder model. The subAbeta rat model of AD was induced by continuous i.c.v. infusion of 160 pmol/day Abeta(1-42) via a 14-day osmotic pump. Behavioral tests and electrophysiological recordings showed that subAbeta rats were not significantly different from control rats. However, chronically stressed subAbeta rats showed more significant impairment of cognitive functions and early-phase LTP (E-LTP) than that caused by stress alone. Molecular analysis of essential signaling molecules after induction of E-LTP revealed an increase in the levels of p-CaMKII in control as well as subAbeta infused rats, but not in stressed or stressed at-risk rats. In addition, compared to unstimulated control, the levels of both total CaMKII and calcineurin were increased in all stimulated animals groups after HFS. These findings suggest that the stress-induced alterations may accelerate the impairment of cognition and synaptic plasticity in individuals "at-risk" for AD.

Glomerular type 1 angiotensin receptors augment kidney injury and inflammation in murine autoimmune nephritis.

Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.