Intranasal CEPO-FC prevents attention deficits in streptozotocin-induced rat model of Alzheimer's disease: Focus on synaptic plasticity-related factors.

Alzheimer's disease remains an issue of great controversy due to its pathology. It is characterized by cognitive impairments and neuropsychiatric symptoms. The FDA approved medications for this disease, can only mitigate the symptoms. One reason for the lack of effective medications is the inaccessibility of the brain which is encompassed by the blood-brain barrier, making intranasal (IN) route of administration potentially advantageous. Male Wistar rats underwent stereotaxic surgery to induce an Alzheimer's disease model via intracerebroventricular (ICV) streptozotocin injection, and Carbamylated Erythropoietin-Fc (CEPO-FC), a derivative of Erythropoietin without its harmful characteristics, was administered intranasally for ten consecutive days. Cognition performance for memory and attention was assessed using the Novel Object Recognition Test and the Object-Based Attention Test respectively. Depression like behavior was evaluated using the Forced Swim Test. Western blotting was done on the extracted hippocampus to quantify STIM proteins. Calbindin, PSD-95, Neuroplastin, Synaptophysin and GAP-43 genes were assessed by Realtime PCR. Behavioral tests demonstrated that IN CEPO-FC could halt cognition deficits and molecular investigations showed that, STIM proteins were decreased in Alzheimer's model, and increased after IN CEPO-FC treatment. Calbindin and PSD-95 were downregulated in our disease model and upregulated when treated with IN CEPO-FC. While Neuroplastin, and GAP-43 expressions remained unchanged. This study suggests that IN CEPO-FC in low doses could be promising for improving cognition and synaptic plasticity deficits in Alzheimer's disease and since IN route of administration is a convenient way, choosing IN CEPO-FC for clinical trial might worth consideration. See also the graphical abstract(Fig. 1).

Combined use of hair follicle stem cells and CEPO (carbamylated erythropoietin)-Fc in a rat model of chronic cerebral hypoperfusion: A behavioral, electrophysiological, and molecular study.

BACKGROUND: In dementia, synaptic dysfunction appears before neuronal loss. Stem cell therapy could potentially provide a promising strategy for the treatment of dementia models. The carbamylated erythropoietin fusion protein (CEPO-Fc) has shown synaptotrophic effects. This study aimed to determine the efficiency of the combined use of hair follicle stem cells (HFSC) and CEPO-Fc in the basal synaptic transmission (BST) and long-term plasticity (LTP) of chronic cerebral hypoperfusion (CCH) rats. METHODS: We divided 64 adult rats into control, sham, CCH+vehicle, CCH+CEPO, CCH+HFSC, and CCH+HFSC+CEPO groups. The CEPO-Fc was injected three times/week for 30 days. HFSC transplantation was done on days 4, 14, and 21 after surgery. The Morris water maze test and passive avoidance were used to assess memory. BST and LTP were assessed by a field-potential recording of the CA1 region. The hippocampal mRNA expression of IGF-1, TGF-ß1, ß1-Catenine, NR2B, PSD-95, and GSk-3ß was evaluated by quantitative RT-PCR. RESULTS: Following combination therapy, spatial memory retention, and BST showed significant improvement relative to HFSC and CEPO-Fc groups. These effects were also confirmed by recovered mRNA expression of ß1-catenin, TGF-ß1, and NR2B. GSK-3ß expression was downregulated in all treatment groups. The upregulated PSD-95 was identified in HFSC and combination groups compared to the vehicle group. CONCLUSIONS: These findings indicate that the combined use of HFSC and CEPO-Fc may be more advantageous for treating memory disruption in the CCH model than CEPO-Fc or HFSC alone. This type of combination therapy may hopefully lead to a new approach to treatment for dementia.

Dipeptide mimetic of BDNF ameliorates motor dysfunction and striatal apoptosis in 6-OHDA-induced Parkinson's rat model: Considering Akt and MAPKs signaling.

Parkinson's disease (PD) is a progressive and debilitating neurodegenerative disorder associated with motor and non-motor complaints. Dysregulation of neurotrophic factors and related signaling cascades have been reported to be common events in PD which is accompanied by dopaminergic (DA) neuron demise. However, the restoration of neurotrophic factors has several limitations. Bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylenediamide (BHME) is a dipeptide mimetic of brain-derived neurotrophic factor (BDNF) with reported anti-oxidant and neuroprotective effects in several experimental models. The current study has investigated the effect of BHME on 6-hydroxydopamine (6-OHDA)-caused motor anomalies in Wistar rats. In this regard, rats were treated daily with BHME (0.1 or 1 mg/kg) 1 h after 6-OHDA-caused damage until the twelfth day. Afterwards, motor behavior and DA neuron survival were evaluated via behavioral tests and immunohistochemistry (IHC) staining, respectively. Moreover, the activity of Akt, mitogen-activated protein kinases (MAPKs) family, and Bax/Bcl-2 ratio were evaluated by Western blotting. Our results indicated that BHME prevents motor dysfunction and DA cell death following 6-OHDA injection, and this improvement was in parallel with an enhancement in Akt activity, decrement of P38 phosphorylation, along with a reduction in Bax/Bcl-2 ratio. In conclusion, our findings indicated that BHME, as a mimetic of BDNF, can be considered for further research and is a promising therapeutic agent for PD therapy.

Prenatal Methamphetamine Hydrochloride Exposure Leads to Signal Transduction Alteration and Cell Death in the Prefrontal Cortex and Amygdala of Male and Female Rats' Offspring.

In the last decade, there has been a great increase in methamphetamine hydrochloride (METH) abuse by pregnant women that exposes fetus and human offspring to a wide variety of developmental impairments that may be the underlying causes of future psychosocial issues. Herein, we investigated whether prenatal METH exposure with different doses (2 and 5 mg/kg) could influence neuronal cell death and antioxidant level in the different brain regions of adult male and female offspring. Adult male and female Wistar rats prenatally exposed to METH (2 or 5 mg/kg) and/or saline was used in this study. At week 12, adult rats' offspring were decapitated to collect different brain region tissues including amygdala (AMY) and prefrontal cortices (PFC). Western blot analysis was performed to evaluate the apoptosis- and autophagy-related markers, and enzymatic assay was used to measure the level of catalase and also reduced glutathione (GSH). Our results showed that METH exposure during pregnancy increased the level of apoptosis (BAX/Bcl-2 and Caspase-3) and autophagy (Beclin-1 and LC3II/LC3I) in the PFC and AMY areas of both male and female offspring's brain. Also, we found an elevation in the GSH content of all both mentioned brain areas and catalase activity of PFC in the offspring's brain. These changes were more significant in female offspring. Being prenatally exposed to METH increased cell death at least partly via apoptosis and autophagy in AMY and PFC of male and female offspring's brain, while the antioxidant system tried to protect cells in these regions.

Valine feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells.

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity. KEY POINTS: • Valine feeding can mitigate the negative impacts of ammonia on CHO cell growth. • Valine addition assists the ammonia removal mechanism by enriching the TCA cycle. • Ammonia is removed from the media through alanine excretion in valine-fed culture.

Carbamylated erythropoietin improves recognition memory by modulating microglia in a rat model of pain.

Patients with chronic pain often complain about memory impairments. Experimental studies have shown neuroprotective effects of Carbamylated erythropoietin (Cepo-Fc) in the treatment of cognitive dysfunctions. However, little is currently known about its precise molecular mechanisms in a model of inflammatory pain. Therefore, this study aimed to investigate neuroprotective effects of Cepo-Fc against cognitive impairment induced by the inflammatory model of Complete Freund's Adjuvant (CFA). Carbamylated erythropoietin was administrated Intraperitoneally (i.p) on the day CFA injection, continued for a 21-days period. After conducting the behavioral tests (thermal hyperalgesia and novel object recognition test), western blot and ELISA were further preformed on days 0, 7, and 21. The results of this study indicate that Cepo-Fc can effectively reverse the CFA induced thermal hyperalgesia and recognition memory impairment. Additionally, Cepo-Fc noticeably decreased the hippocampal microglial expression, production of hippocampal IL-1ß, and hippocampal apoptosis and necroptosis induced by the inflammatory pain. Therefore, our findings suggest that neuroprotective effects of Cepo-Fc in the treatment of pain related recognition memory impairment may be mediated through reducing hippocampal microglial expression as well as IL-1ß production.

Retraction notice to "Noopept; a nootropic dipeptide, modulates persistent inflammation by effecting spinal microglia dependent Brain Derived Neurotropic Factor (BDNF) and pro-BDNF expression throughout apoptotic process" [Heliyon (2021) e06219].

[This retracts the article DOI: 10.1016/j.heliyon.2021.e06219.].

Noopept; a nootropic dipeptide, modulates persistent inflammation by effecting spinal microglia dependent Brain Derived Neurotropic Factor (BDNF) and pro-BDNF expression throughout apoptotic process.

There are largely unknown associations between changes in pain behavior responses during persistent peripheral inflammation and spinal cell alteration such as apoptosis. Some evidence suggests that microglia and microglia related mediators play notable roles in induction and maintenance of central nervous system pathologies and inflammatory pain. By considering those relationships and microglia related nootrophic factors, such as the Brain Derived Neurotrophic Factor (BDNF) in CNS, we attempted to assess the relationship between microglia dependent BDNF and its precursor with pain behavior through spinal cell apoptosis as well as the effect of Noopept on this relationship. Persistent peripheral inflammation was induced by a single subcutaneous injection of Complete Freund's Adjuvant (CFA) on day 0. Thermal hyperalgesia, paw edema, microglial activity, microglia dependent BDNF, pro-BDNF expression, and apoptosis were assessed in different experimental groups by confirmed behavioral and molecular methods on days 0, 7, and 21 of the study. Our findings revealed hyperalgesia and spinal cell apoptosis significantly increased during the acute phase of CFA-induced inflammation but was then followed by a decrement in the chronic phase of the study. Aligned with these variations in spinal microglial activity, microglia dependent BDNF significantly increased during the acute phase of CFA-induced inflammation. Our results also indicated that daily administration of Noopept (during 21 days of the study) not only caused a significant decrease in hyperalgesia and microglia dependent BDNF expression but also changed the apoptosis process in relation to microglia activity alteration. It appears that the administration of Noopept can decrease spinal cell apoptosis and hyperalgesia during CFA-induced inflammation due to its direct effects on microglial activity and microglia dependent BDNF and pro-BDNF expression.

Efficient refolding of recombinant reteplase expressed in Escherichia coli strains using response surface methodology.

Reteplase is a deleted variant of human tissue plasminogen activator with a complex structure containing nine disulfide bonds. Reteplase is expressed as inclusion bodies in Escherichia coli and needs the additional step of refolding for activation. In this study an experimental design was performed to find the optimal refolding condition for reteplase. The influence of 14 chemical additives was assessed by one factor at a time method and then Taguchi design followed by response surface methodology was employed to find compounds with most significant effects on reteplase refolding and their optimum concentration. We found that 0.13 M histidine, 1.64 M methionine, 0.33 M cysteine, and 0.34 M arginine in addition to the GSH/GSSG is the optimal condition for refolding of reteplase. We also investigated the refolding yield for inclusion bodies obtained from different E. coli strains and found that BL21 (DE3) has the best recovery yield in comparison to Rosetta-gami and Shuffle T7.

Correction to: CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3ß and ERK signaling pathways.

Unfortunately, the original version of this article contained a mistake in the arrangement of representative cell images in Fig. 2. In this figure, the same representative image for Aß group was mistakenly placed for Aß + LY group. The corrected form of this figure is provided in this correction.

Microglia dependent BDNF and proBDNF can impair spatial memory performance during persistent inflammatory pain.

Inflammatory pain is commonly associated with cognitive impairment. However, its molecular mechanisms are poorly understood. Thus, this study was conducted to investigate the molecular mechanisms of behavioral changes associated with inflammatory pain. Briefly, 36 Wistar rats were randomly divided into two main groups: CFA group treated with 100 µL of Complete Freunds' Adjuvant (CFA) and CFA + Minocycline group treated with 100 µL of CFA+40 mg/kg/day of minocycline). After that, each group was divided into three subgroups based on different time points of the study. The pain was induced using CFA and subsequent behavioral changes (i.e., hyperalgesia and learning and spatial memory) were analyzed by the Morris Water Maze (MWM) task and Radiant Heat. Then, the cellular and molecular changes were assessed using Western Blotting, Immunohistochemistry, and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) techniques. Results of the study indicated that CFA-induced pain impaired spatial learning and memory functions. Studying the cellular changes showed that persistent inflammatory pain increased the microglial activity in CA1 and Dentate Gyrus (DG) regions. Furthermore, an increase was observed in the percentage of TUNEL-positive cells. Also, pro-Brain-Derived Neurotrophic Factor (BDNF)/BDNF ratio, Caspase3, and Receptor-Interacting Protein kinase 3 (RIP3) levels increased in the rats' hippocampus following induction of persistent inflammatory pain. These changes were reversed following the cessation of pain as well as the injection of minocycline. Taking together, the results of the current study for the first time revealed that an increase in the microglia dependent proBDNF/BDNF ratio following persistent inflammatory pain leads to cell death of the CA1 and DG neurons that subsequently causes a cognitive deficit in the learning and spatial memory functions.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3ß and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aß25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3ß, and ERK signaling and cell death induced by Aß25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aß25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aß25-35. Additionally, CEPO-Fc noticeably reversed Aß mediated decrement of Akt and GSK-3ß phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aß-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aß25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aß-induced cell toxicity as well as Akt/GSK-3ß and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

Neuroprotective Effect of New Nanochelating-Based Nano Complex, ALZc3, Against Aß (1-42)-Induced Toxicity in Rat: a Comparison with Memantine.

PURPOSE: The current drugs for Alzheimer's disease (AD) are only used to slow or delay the progression of the pathology. So using a novel technology is a necessity to synthesize more effective medications to control this most common cause of dementia. In this study, using nanochelating technology, ALZc3 was synthesized and its therapeutic effects were evaluated in comparison with memantine on a well-known rat model of AD, which is based on Amyloid-ßeta (Aß) injection into the brain. MATERIALS AND METHODS: Aß (1-42) was injected bilaterally into the CA1 area of the hippocampus of male rats and then animals were treated daily by oral administration of Alz-C3, memantine or their vehicles. Activities of antioxidant enzymes catalase and superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) levels, as well as Bax/Bcl-2 ratio, caspase-3 activation, and TNF-α expression were evaluated 7 days after Aß injection. Finally, learning and memory of the rats were assessed by Morris water maze test. RESULTS: ALZc3 and memantine improved memory impairment and antioxidant activity and reduced TNF-α expression, caspase-3 activity and Bax/Bcl-2 ratio in the rat's hippocampus. The results showed a superiority of ALZC3 compared to memantine in reducing caspase-3, increasing CAT activity in Aß (1-42)-injected groups and improving apoptosis factor in healthy mice. CONCLUSION: These results indicated that ALZc3 could significantly prevent the memory impairment and Aß (1-42) toxicity. Thus, ALZc3 could be a promising novel anti-AD agent.

Effect of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) on learning and memory impairment and hippocampal apoptosis induced by intracerebroventricular administration of streptozotocin in rats.

Intracerebroventricular (icv) administration of streptozotocin (STZ) has been used as a metabolic model of sporadic Alzheimer's disease (AD). Erythropoietin (EPO) possesses neuroprotective and memory-improving effects, which might be advantageous in treating different characteristics of AD. Nevertheless, the hematopoietic effect of EPO has hindered its application as a neuroprotective agent. Previous studies have shown that a new Epo derivative called carbamylated Erythropoietin-Fc (CEPO-Fc), yield noticeable neuroprotective effects without affecting hematopoiesis. In this study, the neuroprotective effects of CEPO-Fc on icv-STZ induced memory impairment and hippocampal apoptosis were examined. Adult male Wistar rats weighing 250-300 g were used. STZ was administered on days 1 and 3 (3 mg/kg in divided doses/icv), and CEPO-Fc was administered at the dose of 5000 IU/ip/daily during days 4-14. The animals were trained in Morris water maze during days 15-17, and the memory retention test was performed on the 18th day. Following behavioral studies, the animals were sacrificed and their hippocampi isolated to determine the amounts of cleaved caspase-3 (the landmark of apoptosis). The results showed that CEPO-Fc treatment at the dose of 5000 IU/kg/ip was able to prevent the learning and memory deficit induced by icv-STZ. Western blot analysis revealed that STZ prompted the cleavage of caspase-3 in the hippocampus while pretreatment with CEPO-Fc significantly reduced the cleavage of this protein. Collectively, our findings suggest that CEPO-Fc could restore STZ-induced learning and memory impairment as well as apoptosis in the hippocampal region in a rat model of sporadic AD induced by icv-STZ.

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation.

AIMS: Activated microglia is known as a main mediator of inflammatory pain, but the possible mechanisms of its operation are poorly understood. Microglial cells have considered as one of the main sources of pro-inflammatory cytokines in the CNS. PTEN is one of the important targets of pro-inflammatory cytokines and the main mediator of apoptotic cell death. In this study, we investigated the possible effect of microglial activation on PTEN/PI3K/Akt signaling pathway and apoptosis in an inflammatory rat model of Complete Freund's adjuvant (CFA). METHODS: Persistent peripheral inflammation was induced by a subcutaneous injection of CFA into the rats' right hind paw on day 0. Minocycline (a potent selective inhibitor of microglial) was administered intraperitoneally during days 1-21 after CFA injection. Hyperalgesia was assessed on days 0, 7, and 21 using plantar test, then lumbar spinal cord segments were isolated, and the amount of spinal Iba1 (microglial marker), PTEN, P.Akt, and cleaved caspase-3 (a marker of apoptosis activation) were analyzed using Western blot. The spinal TNF-α levels were assayed by ELISA and the microglia numbers were determined using immunohistochemical technique. RESULTS: Results revealed that increased hyperalgesia was concurrent with an increment of Iba1 (P < 0.001), TNF-α (P < 0.001), PTEN (P < 0.01), cleaved caspase-3 (P < 0.001), and a decrement of P.Akt (P < 0.01) during the acute phase of CFA-induced inflammation, while, at the same time as decreasing hyperalgesia during the chronic phase of study, Iba1 and TNF-α expression significantly decreased and PTEN, cleaved caspase-3, and P.Akt restored to baseline on day 0. Minocycline administration reduced the elevation of spinal Iba1 (P < 0.001), TNF-α (0.001), PTEN (P < 0.01), and cleaved caspase-3 (P < 0.001) expression induced by CFA injection, and also restored Akt activity to the baseline on day 0 (P < 0.001). CONCLUSIONS: These results suggest that microglial-mediated pain following CFA injection might be related in part to increased spinal cell apoptosis which probably is mediated by PTEN/PI3K/Akt deregulation.

Comparative evaluation of the safety and efficacy of recombinant FVIII in severe hemophilia A patients.

OBJECTIVE: This study compared the safety and efficacy of Safacto® versus xyntha® in patients with severe hemophilia A. METHODS: Thirty-three male patients with severe hemophilia A were randomly divided into two groups. Seventeen patients received Safacto® and 16 patients received Xyntha® for four consecutive times. The dosage of FVIII was 40-50 IU/kg for each injection. Plasma level of FVIII activity was evaluated before every injection, 15 minutes after the injection and one month after the start of the trial. The rate of factor VIII activity, pain and joint motion were also assessed before and after the treatment. RESULTS: Plasma level of FVIII clotting activity in Safacto® and Xyntha® were 1.96±0.5 IU/dl and 1.63±0.5 IU/dl and increased to 88.84±25.2 IU/dl and 100.09±17.8 IU/dl, respectively (P<0.001). Pain score and range of motion improvement were 9.3±0.9 and 8.7±0.1 in Safacto® (P=0.17); and 9.4±0.8 and 8.8±0.3 in Xyntha® (P=0.35), respectively. No allergic or other unfavorable reactions was observed with either of the preparations. CONCLUSION: This study showed that Safacto® has a favorable efficacy and safety profile.

CEPO-Fc (An EPO Derivative) Protects Hippocampus Against Aß-induced Memory Deterioration: A Behavioral and Molecular Study in a Rat Model of Aß Toxicity.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease, characterized by extracellular deposition of senile plaques, mostly amyloid ß-protein (Aß) and neuronal loss. The neuroprotective effects of erythropoietin (EPO) have been reported in some models of neurodegenerative disease, but because of its hematopoietic side effects, its derivatives lacking hematopoietic bioactivity is recommended. In this study, the neuroprotective effects of carbamylated erythropoietin-Fc (CEPO-Fc) against beta amyloid-induced memory deficit were evaluated. Adult male Wistar rats weighing 250-300 g were bilaterally cannulated into CA1. Aß25-35 was administered intrahippocampally for 4 consecutive days (5 µg/2.5 µL/each side/day). CEPO-Fc (500 or 5000 IU) was injected intraperitoneally during days 4-9. Learning and memory performance of rats was assessed on days 10-13 using Morris Water Maze, then hippocampi were isolated and the amount of activated forms of hippocampal MAPKs' subfamily, Akt/GSK-3ß and MMP-2 were analyzed using Western blot. From the behavioral results, it was revealed that CEPO-Fc treatment in both 500 and 5000 IU significantly reversed Aß-induced learning and memory deterioration. From the molecular analysis, an increment of MAPKs and MMP-2 activity and an imbalance in Akt/GSK-3ß signaling after Aß25-35 administration was observed. CEPO-Fc treatment prevented the elevation of hippocampal of P38, ERK, MMP-2 activity and also Akt/GSK-3ß signaling impairment induced by Aß25-35 but it had no effect on JNK. It seems that CEPO-Fc prevents Aß-induced learning and memory deterioration, and also modulates hippocampal MAPKs, Akt/GSK-3ß and MMP-2 activity. This study suggests that CEPO-Fc can be considered as a potential therapeutic strategy for memory deficits like AD.

Auto-induction for high level production of biologically active reteplase in Escherichia coli.

Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E. coli. Results showed that using auto-induction strategy at 37 °C, Rosetta-gami (DE3) had the highest level of active and soluble reteplase production in comparison to E. coli strains BL21 (DE3), and Shuffel T7. Temperature dominantly affected the level of active reteplase production. Decreasing the temperature to 25 and 18 °C increased the level of active reteplase by 20 and 60%, respectively. The composition of auto-induction medium also dramatically changed the active production of reteplase in cytoplasm. Using higher enriched auto-induction medium, super broth base including trace elements, significantly increased biologically active reteplase by 30%. It is demonstrated here that auto-induction is a powerful method for expression of biologically active reteplase in oxidative cytoplasm of Rosetta-gami. Optimizing expression condition by decreasing temperature and using an enriched auto-induction medium resulted in at least three times higher level of active reteplase production. Production of correctly folded and active reteplase in spite of its complex structure helps for removal of inefficient and cumbersome step of refolding.