disease that primarily affects the elderly population of over 65 years of age, and is estimated to account for 50 - 60% of the dementia cases.  The prevalence has been found to rise exponentially with age, ranging from 3.0% in patients aged 65 to 74 years to as much as 47.2% in those aged 85 years. [2,3] This condition is characterized by a progressive loss of memory, deterioration of virtually all intellectual functions, increased apathy, decreased speech function, disorientation, and gait irregularities. 60% - 70% of this disease leading to dementia.  The real cause of AD is poorly understood. Accordingly, WHO in 2015, approximately Worldwide, 47.5 million people have dementia and there are 7.7 million new cases every year. In a developing country, AD is a financially costly disease. A possible therapeutic target for AD is 𝛽-amyloid cleavage enzyme (BACE-1) which represents the protease cleaved in the APP degradation process, leading to production of β-amyloid (βA). Thus, the BACE-1 enzyme is very important in drug discovery as a primary therapeutic target for AD.  This occurs as a result of, reduces the β-amyloid production, that become a promising target to control and progression of AD. 
A possible therapeutic target for AD is 𝛽-amyloid cleavage enzyme (BACE-1) which represents the protease cleaved in the APP degradation process, leading to production of β- amyloid (βA). Thus, the BACE-1 enzyme is very important in drug discovery as a primary therapeutic target for AD.  This occurs as a result of, reduces the β-amyloid production, that become a promising target to control and progression of AD. 
Plant secondary substances have been suggested as po- tential alternatives for AD therapy largely because plants constitute a potential source of bioactive chemicals that have been perceived by the general public as relatively safe and often act at multiple and novel target sites [14,15]. These potential new anti-AD products can be applied to humans in the same manner as conventional anti-AD drugs. Much effort has been focused on them as potential sources of commercial anti-AD products for prevention or treatment of AD. BACE-1 or AChE inhi- bitors from plants have been well reviewed [13,16] re- spectively. Recently, plants in the family Zingiberaceae have drawn attention because they contain anti-AD principles [17,18]. The rhizomes of turmeric, Curcuma longa L., are not only important as a spice or flavoring, but they have also been prescribed for indigestion, hepatitis, jaundice, diabetes, atherosclerosis and bacter- ial infection [19-21]. Curcumin, an active ingredient of C. longa, has been proposed to alleviate Aβ toxicity in transgenic human Aβ and human tau flies by reducing the pre-fibrillar/oligomeric species of Aβ .
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that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the bind- ing of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligo- methylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications intro- duced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors.
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With the recent research advances in molecular biology and technology, many credible hypothes- es about the progress of Alzheimer’s disease (AD) have been proposed, among which the amyloid and cholinergic hypotheses are commonly used to develop reliable therapeutic agents. The multi- target-directed ligand (MTDL) approach was taken in this work to develop multi-functional agents, which can mainly serve as dual BACE 1 and AChE inhibitors. Depending on the scaffolds of (+)-(S)- dihydro-ar-tumerone and (−)-gallocatechin gallate, 3 series of new compounds have been designed, synthesized and evaluated, from which we have identified 2-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4- tetrahydroisoquinolin-6-yl) isoindoline-1,3-dione (3d) as a new cholinesterase and β-secretase dual inhibitor without toxicity. Furthermore, 3d also exhibits hydrogen peroxide scavenging ac- tivity which could help to reduce the reactive oxygen species (ROS) in the brain of AD patients.
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ACE: Angiotensin converting enzyme; AD: Alzheimeir disease; AGE: Advanced glycosilation end product; AMPK: Adenosine monophosfatase protein kinase; BACE 1: Beta-amyloid cleaving enzyme- 1; BMI: Body muscular index; BP: Blood pressure; CAD: Coronary artery disease; CDK: Cyclin dependent kinase; CHF: Cardiac heart failure; CPR: Complete pathologic response; CREB: cAMP responsive element binding protein; CsA: Cyclosporin A; CVD: Cardiovascular disease; DDPPIV: Dipeptidyl peptidase IV; DM: Diabetes mellitus; DYm: Mitochondrial membrane potential; FPG: Fasting plasma glucose; GDM: Gestacional diabetes mellitus; GFR: Glomerular filtration rate; eGFR: Estimated glomerular filtration rate; GLP-1: Glucagon like peptide 1; HAART: Highly active antiretroviral therapy; HALS: HIV associated lipodystrophy syndrome; HIV: Human imunodeficiency virus; HER-2: Human epidermal growth factor receptor type 2; HF: Heart failure; HOMA- IR: Homeostatic model assessment – insulin resistance; IFG: Impaired fasting glucose; IGT: Impaired glucose tolerance; LKB-1: Liver kinase 1; LSM: Lifestyle modification; MET: Metformin; MG: Metylglyoxal; mTOR: Mammalian target of rapamycin; NF-KB: Nuclear factor kappa beta; NRTIs: Nucleoside reverse transcriptase inhibitors; OGTT: Oral glucose tolerance test; PAI-1: Plasminogen activator inhibitor; PCOS: Policystic ovary syndrome; PIs: Protease inhibitors; PTP: Permeability transition pore; ROS: Reactive oxigen species; SIRT-1: Sirtuin 1; T2DM: Type 2 diabetes mellitus; TCS2: Tuberous sclerosis complex 2; TORC2: Transducer of regulated CREB protein 2; TZP: Triazepinone; UCPs: Uncoupled proteins; VEGF: Vascular endothelial growth factor; Vwf: Von Williebrand fator.
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To identify the subcellular compartment to which BACE is targeted after internalization from the cell surface, we per- formed antibody uptake assays as described above. To detect early endosomes, cells were costained with monoclonal antibod- ies recognizing the early endosome antigen 1 (EEA1) (32). Staining of cells after 15 min showed internalization of BACE into some EEA1-positive compartments. No significant differ- ences in the staining pattern between BACE WT and the phos- phorylation site mutants (S498A and S498D) were detected at this time point, demonstrating that internalized BACE is tar- geted to early endosomal compartments independent of its phosphorylation state (Fig. 8, a–f). After 30 min, internalized BACE WT and BACE S498D were efficiently targeted to jux- tanuclear structures (as described above; Fig. 8), showing less colocalization with the EEA1-positive compartments (Fig. 8, g– k). In contrast, the nonphosphorylated mutant BACE S498A showed less pronounced accumulation in juxtanuclear struc- tures (as described above) but appeared to be retained at least partially in EEA1 positive endosomal compartments (Fig. 8, l and m).
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As expected, minocycline was indeed effective in redu- cing inflammation, as COX-2, iNOS and IL-1b levels were all found to be down-regulated in Tg animals trea- ted with minocycline compared to placebo (Figure 1A- C). After minocycline treatment, the microglial soma size of hippocampal cells appeared to be significantly reduced (Figure 1E and 1G), along with an increase in the complexity of microglial arborization (as illustrated in the representative micrograph in Figure 1E). Both biochemical and morphological findings suggested decreased pro-inflammatory activity. Given the well- known complexity of the microglial phenotype , it is possible that minocycline exerted its effect by switching the microglial cells to a more neuroprotective, M2-like phenotype. Further studies will be required to pinpoint the features of microglia in response to minocycline treatment, using alternative markers such as CD45 or arginase-1 [58-60]. Interestingly, the anti-inflammatory effect of minocycline was specific to the hippocampus and the cortex, a region burdened with intracellular A b - oligomers . In fact, microglial cells of the thalamus, an area largely devoid of A b material at this early age, did not appear to be affected by the treatment (Figure 1F and 1H). This result indicates that minocycline speci- fically interfered with a pathological inflammatory pro- cess dependent on intracellular Ab accumulation.
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4.1.2 Quantifizierung der Proteinexpression von BACE-1 und βAPP im RIPA-Lysat Im nächsten Schritt wurden die Ergebnisse von sieben Wiederholungen des RIPA-Lysat- Experimentes (vier Versuchsdurchführungen mit Gewebeproben des Frontallappens und drei Versuchsdurchführungen mit Gewebeproben des Temporallappens) quantifiziert, berechnet und in einem Diagramm zusammengefasst (siehe Abb. 19) dargestellt. Bezüglich der Trisomie 21-Gehirne war zu erkennen, dass die BACE-1-Proteinmengen im Vergleich zu den Kontrollgehirnen 1,4-fach nichtsignifikant (TT-Test; p=0,76) erhöht waren. Die Proteinexpression von βAPP zeigte sich in den T21-Gehirnen im Vergleich zu den Kontrollen 1,4-fach nichtsignifikant (TT-Test; p=0,53) erhöht. Die Ergebnisse für die AD- Gehirne ließen sich in einer 1,5-fachen nichtsignifikanten (TT-Test; p=0,95) BACE-1- und einer 1,1-fachen nichtsignifikanten (TT-Test; p=0,97) βAPP-Proteinexpressionserhöhung zusammenfassen. In Bezug auf die AD-Gehirne stimmten die Ergebnisse sehr gut mit den bereits publizierten Daten überein (107-112). Man geht davon aus, dass βAPP in Alzheimer-Gehirnen nicht erhöht ist und auch die hier ermittelten Ergebnisse zeigten, dass keine signifikante Erhöhung der βAPP-Proteinexpression in den AD-Gehirnen im Vergleich zu den Kontrollgehirnen vorlag. Außerdem stimmte die in der Literatur angegebene Überexpression von BACE-1 im AD-Gehirn mit dem hier gezeigten Trend zur Erhöhung überein.
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The observation arena, with a size of 45 cm × 35 cm × 20 cm, was made up of white plastic and was located in a testing room that was dimly lit by con- stant illumination at about 40 lx in the testing arena. The objects chosen were 3 identical cuboid blue plastic blocks (A; 4.5 cm × 4.5 cm × 5 cm) and a white cylin- drical plastic bottle (B; height: 7 cm; diameter: 4 cm) that was filled with water. These objects were heavy enough to prevent the mice from moving them. Ac- cording to our screening test, these 2 kinds of objects elicited roughly the same exploratory time relative to the other. Two identical sets of arenas and objects were used by 2 observers who conducted the tests independently in the same room but who were hid- den from each other. Each person randomly tested half of the mice in each group. The test lasted for 3 days. On day 1, the mice were placed in the arena for 10 min in order to reduce neophobic responses and habituate the animals to the stimuli that were pre- sented in the empty arena. On day 2, 2 identical ob- jects (A1A2 or B1B2) were placed at opposite sides in each arena 8 cm from the walls and with 29 cm be- tween them. The mice were placed in the middle of the space between the objects in order to start a 10-min period of object learning and training. On day 3, 1 familiar (previously observed) object and 1 new object were placed in the arena (A3B or B3A for each arena), and the mice were placed in the middle of the space between the objects to start a 10-min object recognition test. Object exploration was defined as the exhibition of the orientation in which the animal’s
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The following potential prognostic factors will be mea- sured in the questionnaires: 1) demographic characteristics e.g. age and gender, 2) characteristics of the compliant e.g. duration of the complaint, the perceived cause, pain response to activity and position, 3) baseline functional disability (RDQ), 4) lifestyle e.g. smoking and alcohol use, 5) comorbidity (Self-administered Comorbidity Question- naire), 6) psychological factors e.g. kinesiophobia, pain cat- astrophizing, back beliefs, expectations of recovery, emotional well-being, 7) work-related factors e.g. physical workload, job satisfaction and co-workers’ support and 8) received treatment due to the back complaints e.g. medi- cation and number of consultations. We will also measure characteristics of the national health system of the differ- ent countries joining the BACE consortium (e.g. insurance form, present guidelines, availability off direct access to medical facilities).
BACE species, which is a multiple of the calculated size of BACE at 60 kDa, the authors term this BACE species a dimer, although they do not strictly show, that this is the case. The BACE “dimer”, was not detected by a pro-domain specific antibody suggesting that it consists of mature BACE molecules. Furthermore, BACE homodimers could only partially be separated by elution form a Q-Sepharose column at high salt conditions. BACE enriched by Ion exchange chromatography was subsequently incubated with the aspartic protease inhibitor pepstatin A-agarose beads, washed and eluted exclusively as a dimer whereas a monomer was detected in the flow through. When the same was done with purified soluble ectodomain of BACE, it was shown that it was more easily dissociated from pepstatin by the washing step, suggesting a lower affinity to the inhibitor pepstatin. Also, the ectodomain appeared exclusively as a monomer on all SDS gels. Schmechel et. al. then went on to analyze a BACE mutant in which one of the two critical aspartates of the active site was replaced by an Alanine (D289A), such that one would expect this mutant to be inactive. Surprisingly, this mutant had similar activity as wtBACE as assayed by the production of soluble APP. To explain this, the authors suggest that in vivo, the active site of the β-secretase could be composed of two N- terminal DTGS active site motifs of two separate BACE molecules which cooperate as a dimer, so that one missing Aspartate within the monomer, as is the case in D289A could be compensated by dimerization. The authors furthermore speculate that although BACE has two active site motifs, it may have adopted the mechanism of the single lobe retroviral proteases that constitute their active site upon dimerization of two lobes only containing one half of the actives site. Also dimerization might be a means to regulate enzymatic activity 235 .
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The 7th variable, i.e. y d t−1 (GDP excess demand), is lowly probable (PIP < 1/3). Instead of y d t−1 we obtained ∆ n t−1 (nominal National Debt), which is classified as medium probable variable (1/3 ≤ PIP < 2/3). We can find a possible explanation of this discrepancy using jointness analysis. According to jointness results, variable y t−1 d is a strong substitute for ∆ n t−1 with value of J LS (y d
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Wild type (IFN-γ+/+) and IFN-deficient (IFN-γ-/-) mice were both highly susceptible to the development of AIA. One day after intra-articular (i.a.) administration of methylated bovine serum albumin (mBSA) we detected small but significant (P < 0.05) increases in plasma levels of IL-1β in IFN-γ-/- mice (13.4 ± 1.1 pg/ml vs. 8.7 ± 1.3 pg/ml for WT). IFN-γ levels were only detectable in WT mice (36.8 ± 0.4 pg/ml) (Figure 4A). IL-1β nor IFN-γ were detectable in plasma samples at subsequent time points (Day 2 and Day 3). We observed moderate IL-1β expres- sion in joints affected by AIA on Day 1. Abundant stain- ing was detected in the synovial lining layer, joint exudate and focal areas of synovial infiltration; it was comparable in WT and IFN-γ-/- mice (Figures 4B, C). Histological evaluation of joint tissue specimens demonstrated that all inflammatory parameters were equivalent; this was reflected in the Arthritis Index (AI) which was not signif- icantly different in WT and IFN-γ-/-; 6.4 ± 0.4 vs. 7.0 ± 0.6 (Figure 4D). There was no evidence of cartilage deple- tion by Safranin-O/fast green staining despite the dis- cernible inflammatory synovial environment (Figure 4E- H).
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feasibility and acceptability qualitatively while quanti- tative information will be used for the design of the full trial. Second, qualitative and quantitative will be assessed generally during the same intervals of the study. Focus group discussions will be conducted pre- and post-trainings around the timing of pre- and post-quantitative assessments. Similarly, follow-up qualita- tive and quantitative assessments will occur after a few months of practice and 1 year later. Third, both methods will be clearly documented in publications with regard to sampling, data collection, and analysis. Fourth, integra- tion will occur in regard to health workers qualitative descriptions of their stigma, knowledge, and compe- tency, which will be integrated with quantitative scores on these three variables. Fifth, because this is a pilot study, inference testing on the quantitative data are limited; therefore, we cannot compare qualitative and quantitative data with regard to effectiveness of the RESHAPE program. Sixth, insights resulting spe- cifically from integration of qualitative and quantita- tive findings will be highlighted.
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A total of 300 people were confirmed to have SMD by the psychiatric nurses. See Fig. 1 for a flow chart of identifica- tion, referral, attendance and confirmation of SMD. Two people with possible SMD refused to participate in pro- ject assessments. Six people with established diagnoses of SMD preferred to continue receiving care from spe- cialist mental health services located in the neighbour- ing district or Addis Ababa. After 6 months of the new service being available, 61 people referred with probable SMD had not accessed PHC-based care. Twelve months after recruitment was closed, an additional 28 people had received treatment for SMD at different PHCs in the district. These 28 people were confirmed to be incident cases that would not have been eligible for treatment when treatment was introduced. Therefore, the contact coverage for SMD was calculated to be 81.3% (300/369). Of those who accessed the service, 257 (85.7%) had a primary psychotic disorder and 43 (14.3%) had bipolar disorder or major depressive disorder with psychotic features.
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XXI blocks genome localization at ND10 but not capsid uptake or disassembly. We sought to determine at which step during the infectious process ␥ -secretase activity is required. There was no obvious effect upon the efficiency of capsid binding and endocytic uptake in the presence of XXI, as determined by immunofluorescence analysis with antibody reactive with the virion surface (data not shown). Upon HPV16 pseudovirion uptake, disassembly of the capsid re- veals otherwise buried epitopes, including one recognized by monoclonal antibody 33L1-7 that is revealed only as virions disassemble in a late endosome/lysosome compartment during infection (2). Treatment with XXI did not impact the uptake of FIG. 1. ␥ -Secretase inhibitor XXI blocks HPV16 and HPV18 infection of 293TT and HaCaT cells. (A) 293TT cells (untreated in A1) were incubated with HPV16 pseudovirus containing a GFP reporter either without (A2) or with (A3) 500 nM XXI. (B) As for panel A, but using HPV18 pseudovirions. (C) As for panel A, but using HaCaT cells. (D) 293TT cells (untreated in D1) were incubated with HPV16 pseudovirus containing a GFP reporter either without (D2) or with the L2-specific and neutralizing monoclonal antibody RG-1 (D3).
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Of all the sequences present in the primary sequence databases, there are few which do not have a structural representative. Two particular examples have come to light very recently which do not have structures and which are of considerable interest in an industrial context,are the sequences of two membrane-bound aspar tic proteases from the human brain. These enzymes have been shown to cleave amyloid precursor protein generating amyloid beta peptide, which is thought to be responsible for the pathology and cognitive decline in Alzheimer’s disease. These sequences, BAE2_HUMAN and BACE HUMAN show a 50% sequence identity to one another, and upon analysis, both exhibit sequence identities of ^25% with most non-viral aspartic proteases in the PDB (highest is 26% for both enzymes with saccharopepsin). Such a sequence relationship has reproduced good structural similarities (see figures 5.17 and 5.18) and it is quite likely th a t such a structural similarity exists between these enzymes and those we have already seen. Indeed, there is considerable identity in the regions hosting the catalytic DTG/DSG motifs. Thus the industrial design and manufacture of an inhibitor for
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AChE: acetylcholinesterase; AD: Alzheimer’s disease; ATC: Anatomical Thera‑ peutic Chemical (Classification System); Aβ: amyloid β; BACE‑1: β‑secretase1; BBB: blood–brain barrier; BuChE: butyrylcholinesterase; Cb: total brain concen‑ tration; Cu,b: unbound brain concentration; CDK: cyclin‑dependent kinase; CNS: central nervous system; CYP: cytochrome P450; EGFR: epidermal growth factor receptor; FDA: Food and Drug Administration; FGFR: fibroblast growth factor receptors; FMPA: N‑(furan‑2‑ylmethyl)‑N‑methylprop‑2‑yn‑1‑amine (a propargylamine inhibitor of MAO); GPCR: G‑protein coupled receptor; H3R: histone type 3 receptor; HBA: hydrogen bond acceptor; HBD: hydrogen bond donor; HCV: hepatitis C virus; HDAC: histone deacetylase; HER2: human epider‑ mal growth factor receptor 2; 5‑HT: serotonin; 5‑HT4R: serotonin receptor type 4; LB‑VS: ligand‑based virtual screening; mAChR: muscarinic M1 acetylcholine receptor; MAO: monoamine oxidase; MoA: mechanism(s) of action; MTD: multi‑target drugs; MTDL: multi‑target designed ligand; NME: new molecular entities; NMR: nuclear magnetic resonance (NMR) spectroscopy; PD: Parkin‑ son’s diseases; PDGFR: platelet‑derived growth factor receptor beta; QSAR: quantitative structure–activity relationship; RET: mast/stem cell growth factor receptor kit (SCFR); ROS: reactive oxygen species; SB‑VS: structure‑based vir‑ tual screening; SDAM: serotonin‑dopamine activity modulator; VEGFR: vascular endothelial growth factor receptors; VS: virtual screening.
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Attenuating Ab production, for instance by inhibiting either of the respective proteases BACE-1 or g-secretase, is considered an attractive strategy for preventing dis- ease progression in patients suffering from Alzheimer ’ s Disease. However, both of these protease inhibition approaches have met several challenges over recent years. BACE-1 has been a difficult target from a chemi- cal tractability point of view with few compounds enter- ing clinical development, most likely due to the difficulties in achieving the combination of necessary enzyme inhibition with adequate brain exposure. More- over, compounds that target g-secretase have been asso- ciated with severe side effects since several other substrates with likely physiological relevance are cleaved by the g-secretase complex. When compound exposure wanes, treatment with g-secretase inhibitors actually results in increased Ab levels, a so called Ab rebound/ rise. Considering these facts we felt it important to investigate whether combining a BACE inhibitor with a g-secretase inhibitor would result in synergistic efficacy A.