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Deposition of amyloid-β (Aβ) is central to Alzheimer's disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aβ deposition including monomeric Aβ40, Aβ42 and Aβ oligomers/protofibrils, Aβ species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N-terminally intact and truncated Aβ40 and Aβ42, as well as Aβ oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life-threatening brain swelling/hemorrhage within one week post-injury. The TBI tissues were compared to post-mortem AD brains (n = 5), to post-mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aβ40 and Aβ42 were not elevated by TBI. The levels of Aβ oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aβ oligomers/protofibrils (P < 0.05) and of both N-terminally intact and truncated Aβ42 (P < 0.05) compared to APOE ε3/4-negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aβ aggregates (commonly referred to as Aβ plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aβ aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short- and long-term outcome in TBI.
Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson's disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1-10) and all C-terminal epitopes (90-140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1-90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1-10 and epitopes between positions 90-140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.
The aggregation of alpha-synuclein (alpha Syn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of aSyn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between aSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human aSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human aSyn, a PLA signal indicating close proximity between aSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human aSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of aSyn and the SNARE proteins in primary neuronal cultures
Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.
BACKGROUND: The amyloid hypothesis in Alzheimer disease (AD) considers amyloid β peptide (Aβ) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated Aβ peptides especially N-truncated pyroglutamate AβpE3-42 have been extensively studied. Together with full-length Aβ1-42 and Aβ1-40, N-truncated AβpE3-42 and Aβ4-42 are major variants in AD brain. Although Aβ4-42 has been known for a much longer time, there is a lack of studies addressing the question whether AβpE3-42 or Aβ4-42 may precede the other in Alzheimer's disease pathology.
RESULTS: Using different Aβ antibodies specific for the different N-termini of N-truncated Aβ, we discovered that Aβ4-x preceded AβpE3-x intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel Aβ4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. While NT4X-167 significantly rescued Aβ4-42 toxicity in vitro no beneficial effect was observed against Aβ1-42 or AβpE3-42 toxicity. Phenylalanine at position four of Aβ was imperative for antibody binding, because its replacement with alanine or proline completely prevented binding. Although amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 PS1Δ9 mutation. A consistent staining was observed in blood vessels in all AD cases with cerebral amyloid angiopathy. There was no cross-reactivity with other aggregates typical for other common neurodegenerative diseases showing that NT4X-167 staining is specific for AD.
CONCLUSIONS: Aβ4-x precedes AβpE3-x in the well accepted 5XFAD AD mouse model underlining the significance of N-truncated species in AD pathology. NT4X-167 therefore is the first antibody reacting with Aβ4-x and represents a novel tool in Alzheimer research.
The hallmarks of Alzheimer's disease (AD) are the accumulation of A beta plaques and neurofibrillary tangles composed of hyperphosphorylated tau. We developed sensitive cellular assays using human embryonic kidney-293T cells to quantify intracellular self-propagating conformers of A beta in brain samples from patients with AD or other neurodegenerative diseases. Postmortem brain tissue from patients with AD had measurable amounts of pathological A beta conformers. Individuals over 80 years of age had the lowest amounts of prion-like A beta and phosphorylated tau. Unexpectedly, the longevity-dependent decrease in self-propagating tau conformers occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the ability of postmortem AD brain homogenates to induce misfolded tau in the cellular assays showed an exponential decrease with longevity, with a half-life of about one decade over the age range of 37 to 99 years. Thus, our findings demonstrate an inverse correlation between longevity in patients with AD and the abundance of pathological tau conformers. Our cellular assays can be applied to patient selection for clinical studies and the development of new drugs and diagnostics for AD.
Background: A majority of mutations within the beta-amyloid region of the amyloid precursor protein (APP) gene cause inherited forms of intracerebral hemorrhage. Most of these mutations may also cause cognitive impairment, but the Arctic APP mutation is the only known intra-beta-amyloid mutation to date causing the more typical clinical picture of Alzheimer disease. Objective: To describe features of 1 Swedish and 1 American family with the previously reported Arctic APP mutation. Design, Setting, and Participants: Affected and non-affected carriers of the Arctic APP mutation from the Swedish and American families were investigated clinically. In addition, 1 brain from each family was investigated neuropathologically. Results: The clinical picture, with age at disease onset in the sixth to seventh decade of life and dysfunction in multiple cognitive areas, is indicative of Alzheimer disease and similar to the phenotype for other Alzheimer disease APP mutations. Several affected mutation carriers displayed general brain atrophy and reduced blood flow of the parietal lobe as demonstrated by magnetic resonance imaging and single-photon emission computed tomography. One Swedish case and 1 American case with the Arctic APP mutation came to autopsy, and both showed no signs of hemorrhage but revealed severe congophilic angiopathy, region-specific neurofibrillary tangle pathological findings, and abundant amyloid plaques. Intriguingly, most plaques from both of these cases had a characteristic ringlike character. Conclusions: Overall, our findings corroborate that the Arctic APP mutation causes a clinical and neuropathological picture compatible with Alzheimer disease.
Previous studies identified engulfment adapter phosphotyrosine binding (PTB) domain containing 1 (GULP1) as an NPXY-motif interactor of low-density lipoprotein receptor-related protein 1 (LRP1) and suggested a potential relevance in Alzheimer's disease (AD). Since AD associated proteins amyloid-beta A4 precursor protein (APP) and LRP1 were shown to interact with the PTB domain of Fe65 and several other adapters via their intracellular NPXY-motifs, we examined a possible interaction of GULP1 PTB domain with the YENPTY-motif of APP. Here we demonstrate that GULP1 is present in human hippocampal and neocortical neurons. Confocal live cell imaging revealed that coexpressed and endogenous GULP1 colocalizes with APP in the Golgi and endoplasmic reticulum. Analysis of the interacting domains by co-immunoprecipitation of point and deletion mutants revealed that the interaction depends on the PTB domain of GULP1 and the YENPTY-motif of APP. Coexpression of GULP1 affected APP cell surface localization and suppressed generation of Abeta40/42 and sAPPalpha. Taken together, these data identify GULP1 as a novel neuronal APP interacting protein that alters trafficking and processing of APP.
An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4°C or -24°C.

Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4°C and -24°C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24°C compared to at +4°C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4°C or -24°C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.
BACKGROUND/AIMS: Increased cerebrospinal fluid (CSF) tau, decreased CSF amyloid-beta42 (Abeta42) and the apolipoprotein E gene (APOE) epsilon4 allele predict progression from mild cognitive impairment (MCI) to Alzheimer's disease (AD). Here, we investigated these markers to assess their predictive value and influence on the rate of disease progression. METHODS: Using ELISA, we measured the CSF biomarkers in 47 AD patients, 58 patients with MCI and 35 healthy control subjects. Twenty-eight MCI patients revisited the clinic and half of them progressed to AD during a period of 3-12 years. RESULTS: The expected changes in CSF total (T)-tau, phosphorylated (P)-tau and Abeta42 levels were found in AD, confirming the diagnostic value of these biomarkers. We were also able to corroborate an increased risk for progression from MCI to AD with elevated CSF T-tau and P-tau and with the presence of the APOE epsilon4/epsilon4 genotype, but not with decreased Abeta42. Finally, for the first time we demonstrated that MCI subjects with high CSF T-tau or P-tau and APOE epsilon4 homozygosity progressed faster from MCI to AD. CONCLUSIONS: CSF T-tau and P-tau as well as the APOE epsilon4/epsilon4 genotype are robust predictors of AD and are also associated with a more rapid progression from MCI to AD.
Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2) and v-myc myelocytomatosis viral oncogene homolog (MYC), were increased in Alzheimer's disease (AD) (P < .05). Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis.
The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to β-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of β-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to β-sheet structure that coincides with the formation of α-synuclein oligomers. Albeit through different kinetic pathways depending on the degree of cross-linking.
Rare coding variants in TREM2, PLCG2, and ABI3 were recently associated with the susceptibility to Alzheimer's disease (AD) in Caucasians. Frequencies and AD-associated effects of variants differ across ethnicities. To start filling the gap on AD genetics in South America and assess the impact of these variants across ethnicity, we studied these variants in Argentinian population in association with ancestry. TREM2 (rs143332484 and rs75932628), PLCG2 (rs72824905), and ABI3 (rs616338) were genotyped in 419 AD cases and 486 controls. Meta-analysis with European population was performed. Ancestry was estimated from genome-wide genotyping results. All variants show similar frequencies and odds ratios to those previously reported. Their association with AD reach statistical significance by meta-analysis. Although the Argentinian population is an admixture, variant carriers presented mainly Caucasian ancestry. Rare coding variants in TREM2, PLCG2, and ABI3 also modulate susceptibility to AD in populations from Argentina, and they may have a European heritage.
Point mutations in the amyloid-beta (A beta) coding region produce a combination of mutant and WT A beta isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant A beta determines WT A beta conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of A beta deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT A beta, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant A beta 40 fibrils into transgenic mice expressing only WT A beta induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant A beta 40 prions induce a conformation of WT A beta similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial A beta prion conformations, which kinetically dominate the spread of prions in the brain.
Tau is a microtubule-associated protein that is important for establishing and maintaining neuronal morphology. In addition to its role in normal cells, tau protein is involved in many neurodegenerative diseases, e.g. Alzheimer's disease (AD) and frontotemporal dementia, as the main component of intraneuronal aggregates. Alternative splicing of tau gene in the brain can give rise to at least six protein variants. A causative role of skewed tau exon 10 inclusion has been defined in frontotemporal dementia; however, no link was established between the aberrant splicing of tau and AD. Here, we applied a single-molecule-based technology, polymerase colony or polony, to simultaneously monitor tau splicing variant and haplotype profile in sporadic AD and normal brains. We found that the coordinated expression of tau exons 2 and 10 is altered in AD. Additional investigations of cis and trans mechanisms of this observation revealed a decreased protein expression of a known tau splicing factor, htra2-beta-1 in AD, thereby implicating a trans mechanism. Our results demonstrate that dysregulation of combinatorial splicing might serve as a signature for aging-related diseases, and the polony assay could be widely adapted for the study of other tauopathies. Furthermore, splicing-based therapeutics is an emerging area of drug development, and a well-defined and quantitative assay for monitoring single-gene transcriptome will be relevant for such development.
Mosaic loss of chromosome Y (LOY) is the most common somatic genetic aberration and is associated with increased risk for all-cause mortality, various forms of cancer and Alzheimer's disease, as well as other common human diseases. By tracking LOY frequencies in subjects from which blood samples have been serially collected up to five times during up to 22 years, we observed a pronounced intra-individual variation of changes in the frequency of LOY within individual men over time. We observed that in some individuals the frequency of LOY in blood clearly progressed over time and that in other men, the frequency was constant or showed other types of longitudinal development. The predominant method used for estimating LOY is calculation of the median Log R Ratio of probes located in the male specific part of chromosome Y (mLRRY) from intensity data generated by SNP-arrays, which is difficult to interpret due to its logarithmic and inversed scale. We present here a formula to transform mLRRY-values to percentage of LOY that is a more comprehensible unit. The formula was derived using measurements of LOY from matched samples analysed using SNP-array, whole genome sequencing and a new AMELX/AMELY-based assay for droplet digital PCR. The methods described could be applied for analyses of the vast amount of SNP-array data already generated in the scientific community, allowing further discoveries of LOY associated diseases and outcomes.
Objective: Cerebrospinal fluid (CSF) amyloid beta(42) (A beta(42)), total tau (t-tau) and phosphorylated tau (p-tau) are useful as predictors of conversion from mild cognitive impairment (MCI) to Alzheimer's disease (AD) dementia. However, results are contradictory as to whether these biomarkers reflect the future rate of clinical decline. Methods: This is a retrospective study on 196 patients with AD [mild/moderate AD (n = 72) or AD-MCI (n = 124) at baseline] with a follow-up period of 2-9 years' duration (median 6 years). Lumbar punctures were performed at baseline as a part of the diagnostic procedure. Results: We found an increased risk of rapid cognitive decline defined as a drop in the Mini-Mental State Examination score of = 4 points/year in patients with CSF t-tau concentrations above the median (OR 3.31, 95% CI 1.53-7.16) and CSF p-tau above the median (OR 2.53, 95% CI 1.21-5.26). Patients with CSF t-tau in the highest quartile had a higher risk of dying in severe dementia (HR 4.67, 95% CI 1.16-18.82). Conclusions: In this large AD cohort, we found an association between high levels of CSF t-tau and p-tau and a more aggressive course of the disease, measured as a rapid cognitive decline and a higher risk of dying in severe dementia.
Background: Alteration of various metabolites has been linked to type 2 diabetes (T2D) and insulin resistance. However, identifying significant associations between metabolites and tissue-specific alterations is challenging and requires a multi-omics approach. In this study, we aimed at discovering associations of metabolites from subcutaneous adipose tissue (SAT) and plasma with the volume, the fat fraction (FF) and the insulin sensitivity (Ki) of specific tissues using [18F]FDG PET/MRI.
Materials and Methods: In a cohort of 42 subjects with different levels of glucose tolerance (normal, prediabetes and T2D) matched for age and body-mass-index (BMI) we calculated associations between parameters of whole-body FDG PET/MRI during clamp and non-targeted metabolomics profiling for SAT and blood plasma. We also used a rule-based classifier to identify a large collection of prevalent patterns of co-dependent metabolites that characterize non-diabetes (ND) and T2D.
Results: The plasma metabolomics profiling revealed that hepatic fat content was positively associated with tyrosine, and negatively associated with lysoPC(P-16:0). Ki in visceral adipose tissue (VAT) and SAT, was positively associated with several species of lysophospholipids while the opposite applied to branched-chain amino acids (BCAA) and their intermediates. The adipose tissue metabolomics revealed a positive association between non-esterified fatty acids and, VAT and liver Ki. On the contrary, bile acids and carnitines in adipose tissue were inversely associated with VAT Ki. Finally, we presented a transparent machine-learning model that predicted ND or T2D in "unseen" data with an accuracy of 78%.
Conclusions: Novel associations of several metabolites from SAT and plasma with the FF, volume and insulin senstivity of various tissues throughout the body were discovered using PET/MRI and a new integrative multi-omics approach. A promising computational model that predicted ND and T2D with high certainty, suggested novel non-linear interdependencies of metabolites.
Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of alpha-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
Parkinson's disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein.
INTRODUCTION: Lumbar puncture (LP) is increasingly performed in memory clinics. We investigated patient-acceptance of LP, incidence of and risk factors for post-LP complications in memory clinic populations.
METHODS: We prospectively enrolled 3868 patients (50% women, age 66 ± 11 years, mini mental state examination 25 ± 5) at 23 memory clinics. We used logistic regression analysis using generalized estimated equations to investigate risk factors for post-LP complications, such as typical postlumbar puncture headache (PLPH) and back pain.
RESULTS: A total of 1065 patients (31%) reported post-LP complaints; 589 patients (17%) reported back pain, 649 (19%) headache, of which 296 (9%) reported typical PLPH. Only few patients needed medical intervention: 11 (0.3%) received a blood patch, 23 (0.7%) were hospitalized. The most important risk factor for PLPH was medical history of headache. An atraumatic needle and age >65 years were preventive. Gender, rest after LP, or volume of cerebrospinal fluid had no effect.
DISCUSSIONS: The overall risk of complications is relatively low. If risk factors shown in this study are taken into account, LPs can be safely performed in memory clinics.
Men have a shorter life expectancy compared with women but the underlying factor(s) are not clear. Late-onset, sporadic Alzheimer disease (AD) is a common and lethal neurodegenerative disorder and many germline inherited variants have been found to influence the risk of developing AD. Our previous results show that a fundamentally different genetic variant, i.e., lifetime-acquired loss of chromosome Y (LOY) in blood cells, is associated with all-cause mortality and an increased risk of non-hematological tumors and that LOY could be induced by tobacco smoking. We tested here a hypothesis that men with LOY are more susceptible to AD and show that LOY is associated with AD in three independent studies of different types. In a case-control study, males with AD diagnosis had higher degree of LOY mosaicism (adjusted odds ratio = 2.80, p = 0.0184, AD events = 606). Furthermore, in two prospective studies, men with LOY at blood sampling had greater risk for incident AD diagnosis during follow-up time (hazard ratio [HR] = 6.80, 95% confidence interval [95% CI] = 2.16-21.43, AD events = 140, p = 0.0011). Thus, LOY in blood is associated with risks of both AD and cancer, suggesting a role of LOY in blood cells on disease processes in other tissues, possibly via defective immunosurveillance. As a male-specific risk factor, LOY might explain why males on average live shorter lives than females.
Tobacco smoking is a risk factor for numerous disorders, including cancers affecting organs outside the respiratory tract. Epidemiological data suggest that smoking is a greater risk factor for these cancers in males compared to females. This observation, together with the fact that males have a higher incidence of and mortality from most non-sex-specific cancers, remains unexplained. Loss of chromosome Y (LOY) in blood cells is associated with increased risk of nonhematological tumors. We demonstrate here that smoking is associated with LOY in blood cells in three independent cohorts [TwinGene: odds ratio (OR) = 4.3, 95% CI = 2.8-6.7; ULSAM: OR = 2.4, 95% CI = 1.6-3.6; and PIVUS: OR = 3.5, 95% CI = 1.4-8.4] encompassing a total of 6014 men. The data also suggest that smoking has a transient and dose-dependent mutagenic effect on LOY status. The finding that smoking induces LOY thus links a preventable risk factor with the most common acquired human mutation.
Introduction: Intraneuronal inclusions of alpha-synuclein are commonly found in the brain of patients with Parkinson's disease and other a-synucleinopathies. The correlation between alpha-synuclein pathology and symptoms has been studied in various animal models. In (Thy-1)-h[A30P] alpha-synuclein transgenic mice, behavioral and motor abnormalities were reported from 12 and 15 months, respectively. The aim of this study was to investigate whether these mice also display symptoms at earlier time points.
Methods: We analyzed gait deficits, locomotion, and behavioral profiles in (Thy-1)-h[A30P] alpha-synuclein and control mice at 2, 8, and 11 months of age. In addition, inflammatory markers, levels of alpha-synuclein oligomers, and tyrosine hydroxylase reactivity were studied.
Results: Already at 2 months of age, transgenic mice displayed fine motor impairments in the challenging beam test that progressively increased up to 11 months of age. At 8 months, transgenic mice showed a decreased general activity with increased risk-taking behavior in the multivariate concentric square field test. Neuropathological analyses of 8- and 11-month-old mice revealed accumulation of oligomeric alpha-synuclein in neuronal cell bodies. In addition, a decreased presence of tyrosine hydroxylase suggests a dysregulation of the dopaminergic system in the transgenic mice, which in turn may explain some of the motor impairments observed in this mouse model.
Conclusions: Taken together, our results show that the (Thy-1)-h[A30P] alpha-synuclein transgenic mouse model displays early Parkinson's disease-related symptoms with a concomitant downregulation of the dopaminergic system. Thus, this should be an -appropriate model to study early phenotypes of alpha-synucleinopathies.
Alzheimer's disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer's disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer's disease patients and non-demented controls to identify potential biomarkers for Alzheimer's disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer's disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer's disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.
Background: Alzheimer's disease (AD) is diagnosed based on a clinical evaluation as well as analyses of classical biomarkers: Aβ42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF). Although the sensitivities and specificities of the classical biomarkers are fairly good for detection of AD, there is still a need to develop novel biochemical markers for early detection of AD.
Objective: We explored if integration of novel proteins with classical biomarkers in CSF can better discriminate AD from non-AD subjects.
Methods: We applied ELISA, mass spectrometry, and multivariate modeling to investigate classical biomarkers and the CSF proteome in subjects (n = 206) with 76 AD patients, 74 mild cognitive impairment (MCI) patients, 11 frontotemporal dementia (FTD) patients, and 45 non-dementia controls. The MCI patients were followed for 4–9 years and 21 of these converted to AD, whereas 53 remained stable.
Results: By combining classical CSF biomarkers with twelve novel markers, the area of the ROC curves (AUROCS) of distinguishing AD and MCI/AD converters from non-AD were 93% and 96%, respectively. The FTDs and non-dementia controls were identified versus all other groups with AUROCS of 96% and 87%, respectively.
Conclusions: Integration of new and classical CSF biomarkers in a model-based approach can improve the identification of AD, FTD, and non-dementia control subjects.
Background: Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls. Principal Findings: In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4x10(-6)) and 14 (IGHV1-67 p = 7.9x10(-8)) which indexed novel susceptibility loci. Significance: The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.
Inclusions of intraneuronal alpha-synuclein (-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of -synuclein is a central feature of the disease pathogenesis. Among the different -synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large -synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in -synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of -synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of -synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective -synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.
Aggregated α-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Experimental evidence indicates that α-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of α-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when α-synuclein oligomers were added to monomeric α-synuclein. In contrast, exogenously added α-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing α-synuclein that were treated with the oligomers displayed reduced α-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected α-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of α-synuclein oligomers into the neocortex of α-synuclein transgenic mice did not induce formation of Proteinase K resistant α-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced α-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.
BACKGROUND: Laboratory tests to assist in the diagnosis and monitoring of neuroinflammatory diseases are scarce. The soluble form of the CD27 molecule (sCD27) is shed in high concentrations by activated T cells and can be detected in the cerebrospinal fluid. The aim of this study was to investigate whether CSF quantitation of sCD27 could discriminate between inflammatory and non-inflammatory neurological diseases.
METHODS: The concentration of sCD27 was measured using a commercially available ELISA in 803 well-defined subjects from a study cohort comprised of 338 patients with neuroinflammatory disease, 338 with non-inflammatory neurological disease and 127 controls without neurological disease.
RESULTS: The median value of cerebrospinal fluid sCD27 was 64 pg/mL (IQR 0-200) in controls, 58 pg/mL (IQR 0-130) in patients with non-inflammatory disease and 740 pg/mL (IQR 230-1800) in patients with inflammatory disease. The likelihood ratio of having an inflammatory disease was 10 (sensitivity 74% and specificity 93%) if the sCD27 concentration was >250 pg/mL. In patients with a known inflammatory condition, the likelihood ratio of having an infection was 10 (sensitivity 40% and specificity 96%) if the sCD27 concentration was >2500 pg/mL.
CONCLUSIONS: The likelihood of having an inflammatory neurological condition is increased with elevated concentrations of sCD27 in cerebrospinal fluid. Rapid tests of sCD27 should be developed to assist clinicians in diagnosis of neuroinflammatory disease.
Oligodendrocyte (OL) death may contribute to white matter pathology, a common cause of network dysfunction and persistent cognitive problems in patients with traumatic brain injury (TBI). Oligodendrocyte progenitor cells (OPCs) persist throughout the adult CNS and may replace dead OLs. OL death and OPCs were analyzed by immunohistochemistry of human brain tissue samples, surgically removed due to life-threatening contusions and/or focal brain swelling at 60.6 +/- 75 hours (range 4-192 hours) postinjury in 10 severe TBI patients (age 51.7 +/- 18.5 years). Control brain tissue was obtained postmortem from 5 age-matched patients without CNS disorders. TUNEL and CC1 co-labeling was used to analyze apoptotic OLs, which were increased in injured brain tissue (p < 0.05), without correlation with time from injury until surgery. The OPC markers Olig2, A2B5, NG2, and PDGFR-alpha were used. In contrast to the number of single-labeled Olig2, A2B5, NG2, and PDGFR-alpha-positive cells, numbers of Olig2 and A2B5 co-labeled cells were increased in TBI samples (p < 0.05); this was inversely correlated with time from injury to surgery (r = -0.8, p < 0.05). These results indicate that severe focal human TBI results in OL death and increases in OPCs postinjury, which may influence white matter function following TBI.
Structural variations are among the most frequent interindividual genetic differences in the human genome. The frequency and distribution of de novo somatic structural variants in normal cells is, however, poorly explored. Using age-stratified cohorts of 318 monozygotic (MZ) twins and 296 single-born subjects, we describe age-related accumulation of copy-number variation in the nuclear genomes in vivo and frequency changes for both megabase- and kilobase-range variants. Megabase-range aberrations were found in 3.4% (9 of 264) of subjects ≥60 years old; these subjects included 78 MZ twin pairs and 108 single-born individuals. No such findings were observed in 81 MZ pairs or 180 single-born subjects who were ≤55 years old. Recurrent region- and gene-specific mutations, mostly deletions, were observed. Longitudinal analyses of 43 subjects whose data were collected 7-19 years apart suggest considerable variation in the rate of accumulation of clones carrying structural changes. Furthermore, the longitudinal analysis of individuals with structural aberrations suggests that there is a natural self-removal of aberrant cell clones from peripheral blood. In three healthy subjects, we detected somatic aberrations characteristic of patients with myelodysplastic syndrome. The recurrent rearrangements uncovered here are candidates for common age-related defects in human blood cells. We anticipate that extension of these results will allow determination of the genetic age of different somatic-cell lineages and estimation of possible individual differences between genetic and chronological age. Our work might also help to explain the cause of an age-related reduction in the number of cell clones in the blood; such a reduction is one of the hallmarks of immunosenescence.
Amyloid-beta (A beta) with 40 (A beta 40) and 42 (A beta 42) amino acids, the main components of amyloid plaques in the Alzheimer's disease (AD) brain, can be measured in human cerebrospinal fluid (CSF) and plasma. Whereas CSF A beta 42 is decreased in AD, some studies have reported changed plasma A beta levels in AD and in subjects with mild cognitive impairment (MCI). To this date it is unclear if and how CSF and plasma levels of A beta correlate with each other in healthy individuals, albeit earlier studies on AD patients found no correlation between CSF and plasma A beta. We have measured A beta 40 and A beta 42 in paired CSF and plasma samples from patients with AD (n=39), MCI (n=29) and healthy control subjects (n= 18). We observed a clear correlation between CSF and plasma levels for both A beta 40 and A beta 42 in healthy individuals, whereas no such correlation could be seen for AD or MCI cases. Similarly to other studies we also found low levels of A beta 42 in AD CSF, whereas there were no significant differences in plasma A beta levels between the diagnostic groups. Our findings suggest that the normal equilibrium between CSF and plasma A beta may be disrupted with the initiation of amyloid deposition in the brain.
Background: Increased concentrations of cerebrospinal fluid (CSF) total tau (t-tau) and phosphorylated tau, as well as decreased amyloid-beta 42 peptide, are biomarkers of Alzheimer's disease (AD) pathology, but few studies have shown an association with AD progression rate. We hypothesized that high CSF tau, as a marker of ongoing neurodegeneration, would predict a more aggressive course of AD, using time to nursing home placement (NHP) as the main outcome. Methods: Our sample inlcuded 234 patients with mild cognitive impairment (MCI) due to AD (n = 134) or mild to moderate AD (n = 100) who underwent lumbar puncture at a memory clinic and were followed for 2-11 years (median 4.9 years). Results: Individuals with CSF t-tau in the highest quartile (>= 900 ng/L) had a higher ratio of NHP, both in the total cohort and in patients with MCI only (adjusted HR 2.17 [95 % CI 1.24-3.80]; HR 2.37 [95 % CI 1.10-5.09], respectively), than the lowest quartile. The association between high t-tau levels and future steep deterioration was confirmed in analyses with conversion to moderate dementia (HR 1.66; 95 % CI 1.08-2.56), rapid decline in Mini Mental State Examination score (>= 4-point drop/12 months), and dying in severe dementia as outcomes. Conclusions: To our knowledge, this is the first study to show that high CSF t-tau levels predict early NHP and conversion to moderate dementia in an AD cohort. Selecting patients with high CSF t-tau, indicating more aggressive neurodegeneration and steeper decline, for AD immunotherapy trials might increase the possibility of showing contrast between active treatment and placebo.
Immunotherapy targeting aggregated alpha-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other alpha-synucleinopathies. We have developed alpha-synuclein oligomer/protofibril selective antibodies that reduce toxic alpha-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective alpha-synuclein antibodies, linear epitope monoclonal alpha-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, alpha-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing alpha-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fc gamma receptors were targeted and we then found that blockage of Fc gamma RI and Fc gamma RIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular alpha-synuclein and mediated via Fc gamma receptors. Altogether, our finding lend further support to the belief that alpha-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic alpha-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of alpha-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.
Background: Due to its neurotoxic properties, oligomeric alpha-synuclein (α-syn) has been suggested as an attractive target for passive immunization against Parkinson's disease (PD). In mouse models of PD, antibody treatment has been shown to lower the levels of pathogenic α-syn species, including oligomers, although the mechanisms of action remain unknown. We have previously shown that astrocytes rapidly engulf α-syn oligomers that are intracellularly stored, rather than degraded, resulting in impaired mitochondria.
Methods: The aim of the present study was to investigate if the accumulation of α-syn in astrocytes can be affected by α-syn oligomer-selective antibodies. Co-cultures of astrocytes, neurons, and oligodendrocytes were derived from embryonic mouse cortex and exposed to α-syn oligomers or oligomers pre-incubated with oligomer-selective antibodies.
Results: In the presence of antibodies, the astrocytes displayed an increased clearance of the exogenously added α-syn, and consequently, the α-syn accumulation in the culture was markedly reduced. Moreover, the addition of antibodies rescued the astrocytes from the oligomer-induced mitochondrial impairment.
Conclusions: Our results demonstrate that oligomer-selective antibodies can prevent α-syn accumulation and mitochondrial dysfunction in cultured astrocytes.
In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.
Pathophysiological impairment of the neurovascular unit, including the integrity and dynamics of the blood-brain barrier (BBB), has been denoted both a cause and consequence of neurodegenerative diseases. Pathological impact on BBB drug delivery has also been debated. The aim of the present study was to investigate BBB drug transport, by determining the unbound brain-to-plasma concentration ratio (K-p,K-uu,K-brain), in aged A beta PP-transgenic mice, alpha-synuclein transgenic mice, and wild type mice. Mice were dosed with a cassette of five compounds, including digoxin, levofloxacin (1 mg/kg, s.c.), paliperidone, oxycodone, and diazepam (0.25 mg/kg, s.c.). Brain and blood were collected at 0.5,1, or 3 h after dosage. Drug concentrations were measured using LC-MS/MS. The total brain-to-plasma concentration ratio was calculated and equilibrium dialysis was used to determine the fraction of unbound drug in brain and plasma for all compounds. Together, these three measures were used to determine the Kp,uu,brain value. Despite A beta or alpha-synuclein pathology in the current animal models, no difference was observed in the extent of drug transport across the BBB compared to wild type animals for any of the compounds investigated. Hence, the present study shows that the concept of a leaking barrier within neurodegenerative conditions has to be interpreted with caution when estimating drug transport into the brain. The capability of the highly dynamic BBB to regulate brain drug exposure still seems to be intact despite the presence of pathology. (C) 2017 The Authors. Published by Elsevier Ltd.
Background: According to the modified amyloid hypothesis the main event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid beta-peptide (A beta) within neurons. Additionally to full-length peptides, a great diversity of N-truncated A beta variants is derived from the larger amyloid precursor protein (APP). Vast evidence suggests that A beta(x-42) isoforms play an important role triggering neurodegeneration due to its high abundance, amyloidogenic propensity and toxicity. Although N-truncated and A beta(x-42) species have been pointed as crucial players in AD etiology, the A beta(5-x) isoforms have not received much attention. Results: The present study is the first to show immunohistochemical evidence of A beta(5-x) in familial cases of AD (FAD) and its distribution in APP/PS1KI, 5XFAD and 3xTG transgenic mouse models. In order to probe A beta(5-x) peptides we generated the AB5-3 antibody. Positive plaques and congophilic amyloid angiopathy (CAA) were observed among all the FAD cases tested carrying either APP or presenilin 1 (PS1) mutations and most of the sporadic cases of AD (SAD). Different patterns of A beta(5-x) distribution were found in the mouse models carrying different combinations of autosomal mutations in the APP, PS1 and Tau genes. All of them showed extracellular A beta deposits but none CAA. Additionally, they were all affected by a severe amyloid pathology in the hippocampus among other areas. Interestingly, neither 5XFAD nor APP/PS1KI showed any evidence for intraneuronal A beta(5-x). Conclusions: Different degrees of A beta(5-x) accumulations can be found in the transgenic AD mouse models and human cases expressing the sporadic or the familial form of the disease. Due to the lack of intracellular A beta(5-x), these isoforms might not be contributing to early mechanisms in the cascade of events triggering AD pathology. Brain sections obtained from SAD cases showed higher A beta(5-x)-immunoreactivity in vascular deposits than in extracellular plaques, while both are equally important in the FAD cases. The difference may rely on alternative mechanisms involving A beta(5-x) peptides and operating in a divergent way in the late and early onset forms of the disease.
The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased beta-secretase cleavage of the amyloid-beta (A beta) precursor protein. This leads to abnormally high A beta levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APP(sw) allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APP(sw) or APP(WT). As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted A beta. Next, coding sequences for the APP(sw)-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APP(sw) transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APP(sw) allele both ex vivo and in vivo-and thereby decrease pathogenic A beta. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased A beta.
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-lambda 465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465 lambda in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.


text: January 04, 2011 The Contender's Magnificent 7 A T/C Contender with a bevy of barrels makes a lot of sense. But which chamberings should you choose? Here's Layne's list of seven sensational cartridges that capitalize on the Contender's versatility. The original Contender was tremendously popular, and the newer G2 version (shown here) has several improvements, one being it is easier to open.My crystal ball is often quite accurate, and I say that with all modesty, but there are times when its look into the future is off by a country mile or two. Case in point is the Thompson/Center Contender. Shortly after its introduction in 1967, I handled the gun--I even had a friend who praised the two he had purchased--but prior to my days of metallic silhouette competition, I had no interest in it whatsoever. This was mainly because I considered it to be one of the homeliest and worst handling firearms I had ever picked up. Bluntly put, I figured its future was less than bright. Others in surprising places shared my opinion. While the company's founder, Warren Center, was working on the design of the Contender, some of his employees thought he was wasting valuable time. Talk about us all being wrong. Contender sales started slowly, but its popularity among hunters and shooters grew as each new option was introduced by T/C. It went on to enjoy great success primarily because each new option increased its versatility far beyond other handgun designs. With its interchangeable-barrel capability and a hammer design that allowed both rimfire and centerfire cartridges to be used, the Contender was like nothing anyone had previously seen. On top of all that, it was affordable, and it proved to be extremely accurate. In other words, the Contender was the right idea in the right place at the right time.The Contender has to be considered one of the all-time great firearms designs simply because it is capable of being so many different things to so many different people with so many different interests. A single Contender has the capability of serving as an entire battery of firearms at far less cost than for a battery made up of other types of guns. Caliber options in handgun, carbine, and rifle barrels range from .17 Mach II and .22 Long Rifle to .44 Magnum and .45-70 Government. The rifle can also be a .410 shotgun or a .45-caliber muzzleloader. J.D. Jones, who owns SSK Industries, also deserves some of the credit for making the Contender successful. That's because as far back as the 1970s he was offering custom barrels in not only factory chamberings unavailable from T/C at the time--the .45-70 being an example--but also in an extensive line of JDJ wildcats designed specifically for the Contender. The present G2 version of the Contender is an improvement over the original in several ways. At the top of my list in importance is the fact that the hammer can be lowered and then recocked without having to break open and then close the barrel. The G2 is also easier to open, and there is a bit more clearance between the front of its grip and the rear of its trigger guard. The latter feature is especially nice when shooting a hard-kicking cartridge. Except for the 209x45 muzzleloader barrel, which is compatible only with the G2, most barrels are interchangeable between old and new Contenders. But grips and buttstocks differ between the two.I bought my first Contender in 1969, but I did not begin to take it seriously until I bought a 10-inch barrel in .30-30 and used it in production-class metallic-silhouette competition. I stuck with the .30-30 in that game for a couple of years before switching to the 7mm TCU. Since those days, I have worked with Contender pistols and rifles in most of the chamberings offered by T/C as well as many of the wildcats offered by SSK. As you might expect, several chamberings have become my favorites. Here are what I consider to be the "Magnificent Seven" centerfire cartridges for T/C's versatile Contender. The .309 JDJ is one of Layne's all-time favorite Contender cartridges. He took this Alaskan caribou at 150 yards with the cartridge loaded with Nosler's 165-grain Ballistic Tip..223 RemingtonWhen it comes to picking off varmints at long range with a Contender, my favorite cartridges are the .17 Remington, the .204 Ruger, and the .223 Remington. The .17 and the .204 shoot a bit flatter, but barrels chambered for them have to be cleaned more often during hot-barrel critter shoots. That's the reason the .223 Remington is the one I choose most often. While 40- and 45-grain bullets exit the muzzle at higher velocities, I find that any good 50-grain bullet at 3,000 fps bucks wind a bit better and is more effective on coyotes. It shoots flat enough, too. When a high-ballistic-coefficient bullet of that weight--such as the Sierra BlitzKing, Nosler Ballistic Tip, or Hornady V-Max--is zeroed 2 inches high at 100 yards, it is good for a dead-on hold on prairie dogs out to about 250 yards, and with the intersection of the crosshairs plastered on the head of a groundhog standing 300 yards away, the bullet will land somewhere in its boiler room.As a Contender cartridge, the .223 Rem. has a couple of other things going for it. It is capable of shooting inside 1/2 MOA in a good barrel, and its recoil is so light you can bump off varmints with it all day long, day after day, and never get enough.6.5 JDJThe 6.5 JDJ is one of those rare cartridges that delivers a level of performance that seems all out of proportion to its size, appearance, and recoil. This mild-mannered little number has been used to take Rocky Mountain elk and even African antelope as large as sable and greater kudu, but it is seen at its best when used on deer-size game. It delivers a deadly blow on game, yet its mild recoil makes it the kind of cartridge you cannot shoot enough. Developed by Jones, who I like to describe as the P.O. Ackley of wildcats for handguns, it is the .225 Winchester case necked up and fireformed to the improved configuration with minimum body taper and a 40-degree shoulder angle. Why the .225 Win. case? According to Jones, its great strength and toughness enables it to survive many full-power firings. And he is correct about that; the 40 cases I started using about 15 years ago are still going. Jones has also said that the powder capacity of the improved .225 Win. case is optimum for 6.5mm caliber in a 14-inch barrel, and its rim makes it a better choice for the Contender than a rimless case.There is no law against using bullets of other weights in the 6.5 JDJ, but the traditional weight when it is used on deer is 120 grains. There was a time when the Speer 120-grain spitzer was the single most popular bullet among hunters, and it is still a good one, but the Nosler Ballistic Tip of the same weight has now stolen much of its thunder. Other excellent choices for this cartridge include Nosler's 125-grain Partition, Swift's 120-grain A-Frame and 130-grain Scirocco, and Hornady's 129-grain SST. When exiting a 14-inch barrel at 2,400 to 2,500 fps and zeroed 3 inches high at 100 yards, any of those bullets is about dead-on point of aim at 200 yards and half the body depth of a deer low at 300 yards, where it is packing close to 1,000 ft-lbs of punch. 7mm TCUThere was a time when handgun metallic-silhouette matches were quite popular in my neck of the woods, and I participated in them quite often. When competing in the unlimited class, I used a custom XP-100 chambered for a wildcat called the 7mm TNT. And at first, I used a 10-inch T/C Contender in .30-30 in production class. I switched to the 7mm TCU for production class immediately after T/C introduced that chambering. Only a few months after getting a 10-inch barrel in 7mm TCU, I used it in the South Carolina state championship and shot it quite well there. The load I used--27.0 grains of H335 behind the Sierra 160-grain spitzer boattail--toppled targets with aplomb, including hard-set rams. I bring all of this up simply because if not for metallic silhouette competition, I might never have gotten around to becoming a fan of the Contender pistol.Formed by necking up the .223 Rem. case and fireforming to minimum body taper and a 40-degree shoulder angle, the 7mm TCU is great fun to shoot due to its extremely mild recoil. While seen at its best as a metallic silhouette cartridge, it will work on deer at relatively close ranges when loaded with the right bullet. Best bullets for deer in this cartridge are the Nosler 120-grain Ballistic Tip, Hornady 120-grain SSP (Single Shot Pistol), and Sierra 130-grain SSP loaded to maximum velocities in a barrel no shorter than 14 inches. There are better deer cartridges for the Contender, but as I have proven to my own satisfaction, the 7mm TCU works about as well as the 7.62x39mm Russian..30-30 WinchesterWhat can I say about the good old .30-30 Winchester that has not already been said many times through the years? As standard chamberings presently offered by T/C go, I consider it to be one of if not the best choice for use on deer-size game. It is accurate, shoots flat enough for a dead-on hold out to 250 yards, and delivers enough punch to down deer-size game at that distance. Case life is also higher in the Contender than in lever-action rifles such as the Winchester 94 and Marlin 336.I have bagged several whitetail deer with a 14-inch barrel in .30-30, and one of the best bucks I have taken to date fell victim to a Contender rifle with a 23-inch barrel. When hunting deer with the rifle version, I usually stick with any good 150-grain spitzer loaded to 2,300 fps, with the Hornady SST being one of my favorites. But due to the lower velocity of the 14-inch barrel, I drop back in bullet weight to the Sierra 135-grain SSP or the Nosler 125-grain Ballistic Tip when using that barrel length. I have also used those two on pronghorn antelope, and they both worked great..309 JDJAnother of my trio of favorite Jones creations is the .309 JDJ, or .30-30 Super as it could just as well have been called. Despite its name, it uses the same .308-inch bullets as the .30-06, .308 Winchester, and other cartridges of the same caliber. All things being equal--barrel length and the chamber pressures to which the two are loaded--it is, depending on bullet weight, 400 to 700 fps faster than the .30-30 Win. Even though the .309 JDJ is customarily loaded to lower chamber pressure than the .308 Win., its 13 percent greater case capacity enables it to tread closely on the heels of that cartridge in performance. I have a custom XP-100 with a 15-inch barrel in .308 Win., and when both are loaded to maximum with a 165-grain bullet, the .309 is only about 100 fps slower in my 14-inch Contender.The .309 JDJ case is formed by necking down .444 Marlin brass and giving it a 40-degree shoulder angle. .375 JDJLike other JDJ cartridges, the case of the .375 JDJ is formed by necking down the .444 Marlin case. My guess is it has been used to take more African game up to elephant, hippo, and Cape buffalo in size than any other cartridge designed for use in a handgun. But it is also an excellent choice for hunters who go after North American game such as elk and moose. There was a time when the Hornady 270-grain Spirepoint was the hands-down favorite of most who hunted with this cartridge, and while it is still an excellent choice, it now has a bit of competition from a couple of bullets from Nosler: the 260-grain AccuBond and the Partition of the same weight. When either bullet exits the muzzle of a 14-inch barrel at 2,000 fps and is zeroed 3 inches high at 100 yards, it lands about 2 inches below line of sight at 200 yards. At that distance, it delivers around 1,600 ft-lbs of energy. If that's not 200-yard elk medicine from a handgun, I don't know what is.If I had to choose between the .375 and the 6.5 JDJs for my next deer hunt, I would head to the woods with the 6.5 JDJ. But for both deer and larger game, the .375 JDJ is the logical choice. Three options in deer loads stand out in this cartridge. For woods hunting where shots are usually inside 100 yards, the Sierra 200-grain FN and the Hornady 220-grain FN--both made for use in the .375 Winchester cartridge--are excellent choices. For hunting deer-size game in open country where shots can be farther out, the Hornady 220-grain Spirepoint is tough to beat. It is also the bullet I would choose for all-around use when hunting deer or black bear in both wooded and open country..45-70 GovernmentThe .45-70 Government has been a favorite of mine since the 1950s. That was when I horse-traded for a Trapdoor Springfield in that chambering. Some years later, when the popular thing to do was to build a rifle in that caliber around the Siamese Mauser action and handload it to velocities that were not far behind those possible with the .458 Win. Mag., I again fell in love with the .45-70. Two more custom rifles in that caliber--a Marlin 1895 and a Ruger No. 1--came later. Later still was a 14-inch barrel for my Contender. I've shot the .45-70 a lot through the years and doubt if I will ever tire of hearing its fat bullet smack a target or seeing it kick up lots of dust at long range.I now have two Contender barrels in .45-70: the 14-incher I got many years ago and a 10-incher from the T/C Custom Shop. The 14-inch barrel is more comfortable to shoot due to its added weight plus the fact that it wears an Arrestor muzzle brake. I actually find the .45-70 to be more comfortable to shoot in a 14-inch Contender wearing a Pachmayr rubber grip than in either my Marlin or Ruger rifles. This holds especially true when 300-grain bullets are loaded in the neighborhood of 1,600 fps. My picks for use on deer are the 300-grain bullets from Sierra, Nosler, and Hornady.There you have them, my picks for the all-time best cartridges for T/C's Contender. Others might be as good, but I haven't found any to be better. That's why in my book they will always be the "Magnificent Seven." 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subset: commoncrawl


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I did do a reading for a friend that recently had a baby, but I have wondered if my interpretation was somewhat colored by the fact that I've known her for about 10 years.vervain wrote:You're less likely (I think) to mess with your intuition if it's for people you've never actually met.
I'd like to offer to do readings on here, but I'm nervous about it since I'm so new at it. The fear of being wrong and having someone mad at me for it is kind of a big issue for me. Actually, there have been a couple times that I've done readings for questions asked on here, but I've been too shy to share them.


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Global markets and improvements in technology have changed the way businesses work. For many, staying competitive means capitalizing on the world being a smaller, more tech-savvy place, and enjoying the benefits of outsourcing.
Hiring an overseas workforce to outsource certain business functions can lead to:
- lower overheads;
- increased growth; and
- greater focus and service quality.
Combined, these benefits mean a happier looking bottom line.
On paper, it all sounds great. But for a business sitting on the fence about whether to offshore, promises and benefits that "sound great" aren't enough.
You need evidence.
Well, the good news is you've come to the right place. The ultimate list of outsourcing statistics puts the proof in the pudding. Using current data from trusted sources, here's why your small business might outsource roles to a managed operations provider or a business process outsourcing (BPO) provider.
Why businesses outsource
As previously mentioned, outsourcing allows companies to lower their overheads. In fact, according to Deloitte's 2016 Global Outsourcing Survey, it's the main reason businesses hire overseas teams.
59% of businesses surveyed said outsourcing was used as a cost-cutting tool. But investing in outsourcing is more than just a money-saving exercise. If we look at all of the responses they suggest outsourcing improves business performance and, therefore, results in better user experience.
Interestingly, 57% of respondents said outsourcing meant they could focus on their core business. 31% outsourced specifically to enhance service quality, while 17% used outsourcing as a way to drive change and disrupt their business norm.
The conclusion is that by outsourcing, businesses are rewarded with extra time and greater staff support. This allows them to scale, remain competitive, and (potentially) improve their services and offerings. That's good news for consumers.
Deloitte's 2016 study of why businesses outsource remains the most relevant and reliable list. But delve deeper into the statistics and it's clear the outsourcing market is far from static.
Offshore popularity is rising
Spending on outsourced services in the global market has grown in the last two decades, from US$45.6 billion in 2000 to US$86.6 billion in 2018. Almost double! And it's not slowing down.
Businesses are feeling positive about their outsourcing relationships. As a result, 57% of US companies increased their use of outsourcing.
Which sectors and functions are outsourced the most?
Some business functions and industry sectors lend themselves better to outsourcing than others. This boils down to practical issues around setup and management.
According to Statista and KPMG's 2017 Global IT-BPO Outsourcing Deal Analysis, IT, defense and government, media, health and pharmaceuticals, and retail are among the most popular outsourcing sectors.
Although all of these sectors make strong contributions to the financial growth of the outsourcing market, telecommunications remains one of the top contributors.
ITO deals — which within the context of the KPMG report includes telecommunications — contributed to 65% of the outsourcing deals sealed in 2017.
Why does the telecommunications sector fair so well?
Constant evolution.
Advancements in technologies, more companies moving to the cloud (which we'll discuss in more detail shortly), and increased global mobile usage, all boost the telecommunications sector and offshoring countries that can support its rapid development.
As it stands, American businesses most commonly outsource IT functions. But they outsource a lot of other functions too. When asked about current and future outsourcing levels, respondents revealed they planned to increase the amount of tax, HR and procurement tasks handed to offshore teams.
Finance shows the biggest difference. Currently, US businesses outsource around 39% of finance functions, but wish to push this up to 89%.
Clearly, these statistics prove how successful outsourcing continues to be for modern American businesses. And the rest of the world seems keen to capitalize on the benefits of outsourcing, too.
Adoption and revenue beyond the Americas
America is a major outsourcing region. When professional services company KPMG analyzed outsourcing deals closed in Q1 2017, the US contributed 41.5% of the total deal value.
But Asia Pacific regions and Europe, the Middle East and Africa (EMA), aren't far behind.
An increased willingness to adopt outsourcing in the EMA and Asia Pacific has sent figures upwards.
In the first quarter of 2017, the EMA's contribution to the global outsourcing deal value was a healthy 35% (with 22.07% of deals coming from the United Kingdom). This was up on the last quarter of 2016, when it sat at 27%.
Likewise, Asia Pacific's contribution to global outsourcing deals rose from a mere 16% in the last quarter of 2016, to 23% in Q1 of 2017.
In the same KPMG report, Australia and India were identified as key contributors to the global outsourcing market.
Effects of disruptive automation and cloud working
When it comes to outsourcing, technology brings pros and cons.
A huge pro is the use of cloud-based working. Although there are concerns around data security and implementation, increased connectivity makes offshoring much easier.
The con is technology's impact on jobs. According to Deloitte, more businesses are hoping to adopt Robotic Process Automation (RPA), leading to improvements in speed, precision and quality of the information used to make critical business decisions.
A.T. Kearney's 2017 study titled The Widening Impact of Automation, shows more than 1 million BPO jobs are at risk in the US, India and the Philippines because of the rise of automation technology. It's thought a single automated service replaces four people.
But it's not all doom and gloom. While it may appear cheaper, automation still needs setting up and monitoring: cue the human workforce.
Plus, some jobs simply can't be automated. Healthcare, HR, telecommunications and customer service aren't as robot-friendly, so offshoring continues to rise, despite increases in automation in other areas.
This means businesses can outsource high-level roles to stay lean in a competitive environment.
The Philippines — a top 10 offshoring destination
When it comes to offshoring, the Philippines is one of the top countries in the world.
How a country is ranked has changed. And the criteria plays to the Philippines' strengths. For example, A.T. Kearney took digital competence in consideration to identify the top outsourcing destinations in its Global Services Location Index (GSLI), while the Tholons Services Globalization Index added digital innovation.
The rankings confirm the Philippines is an industry leader in outsourcing.
So what do the statistics suggest it has going for it?
- Great education, with a 97.5% literacy rate and more than 680,000 uni graduates per year (for perspective, Australia has approx 300,000)
- It's the third-largest English speaking country in the world
- It offers approx 70% lower labor costs than Australia
- The economy has grown around 6% each year since 2013
- Manila, in the Philippines, is a top five Digital Nation Super City, according to 2018 Tholones Services Globalization Index.
With excellent communication skills in both oral and written English, the Philippines offers a balanced package of excellent customer service with reduced labor costs. More importantly, the government is very supportive of the offshoring industry, with a nationwide focus on infrastructure and telecommunications investments, especially in the main cities.
The future of outsourcing
This ultimate list of outsourcing statistics shows how popular offshoring continues to be. As technologies and infrastructure continue to evolve, particularly in the Philippines, companies needing to grow and remain competitive in the global market, can't afford to overlook the cost savings and functional benefits of outsourcing.
Let us help you outsource your business
Launched in 2006, MicroSourcing is now one of the biggest managed operations providers in the Philippines. With over 4,000 employees, seven delivery centers, and close to 200 clients, we help build dedicated teams in the Philippines — covering all roles and levels of specialization. Get your offshore team up and running in as little as six weeks. Send us an email today and let's start discussing your plans.


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Web development is game development minus two lines of code
Posted by Elf Sternberg as programming, python, web development
I recently wrote a game, and found it very much like developing a Javascript-based web application. (My wife, who's a journalist with the video game industry beat, insists "it's a toy, not a game. There is a difference." I will defer.)
If you're a web developer who does a lot of Ajax, there is one kind of event in which you're often very interested. Most events describe what just happened, but advanced Ajax and DOM coding often rely more on what just stopped happening. The Image.onload() and the XHR (XMLHTTPRequest) objects' onreadystatechange handlers are interesting because they tell us when something that takes time is done, not when something has begun.
Another, related component of javascript development is setTimeout(). setTimeout is basically on(when clock reaches this point)=somefunc(). It's not worded that way, but eventually you'll wrap your mind around that basic idea. setTimeout() is just another way of shoving something into the list of events. Along with all the objects on the screen, each of which might have one or more events associated with it, there's a general object, the clock, to which you hook up events with setTimeout(). When you understand this, you'll realize the basic truth:
Everything in web application development is event-driven. Everything. The same is true of games.
The gametoy I wrote is Thinksaber, which is basically a re-implementation of MacSaber for Linux-based Thinkpads. It's written in python and the core is pretty straightforward:
idle_channel.play(sounds['idle'][0]) while 1: event = pygame.event.wait() if event.type == idle_channel.get_endevent(): idle_channel.play(sounds['idle'][0]) if event.type == pygame.JOYAXISMOTION: self.make_noise()
This is a very stripped-down version of the actual core, but it's all we need to understand the main point. Lightsabers, when not being swung around, make an ambient menacing hum. I reserved a channel in the pygame sound interface for that, and call play() on it when the gametoy starts up. (Actually, there's a chunk of code in the actual program that also plays the startup sound, too.)
That sound clip is only so long. When it stops, it has to be restarted. That's what this loop does: if the event was a sudden motion of the laptop, call the make_noise method to figure out how fast and in what direction, but if the event was "end of idle sound," restart the idle sound.
I was struck by just how similar this is to monitoring onload, onreadystatechange, and setTimeout. You, the programmer, don't actually have to do the monitoring; these events are delivered to you on an as-needed basis. The only major
difference between game programming and web programming is that in game programming, at least using the PyGame interface (and many others that I've seen), the loop and the dispatcher are explicit. They're only two lines long, but once you know they're there, you start to see their presence under the hood of IE and Firefox.
- Tags: game programming, games, pygame, python, web programming


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U.S. Department of Justice
Recent News About U.S. Department of Justice View More
Ex-Tallahassee city commissioner indefinitely suspended from law practice following fraud plea
TALLAHASSEE (Florida Record) — Longtime Tallahassee attorney and former City Commissioner Scott Maddox has been indefinitely suspended following an Aug. 15 Florida Supreme Court order and his guilty plea earlier in August regarding influence peddling and corruption allegations, according to a recent announcement by The Florida Bar.
Imprisoned Hillsboro Beach attorney disbarred following 'pump-and-dump' conviction
TALLAHASSEE — Longtime Hillsboro Beach attorney James M. Schneider, sentenced in February for his alleged role in a "pump-and-dump" securities fraud scheme, has been disbarred following a March 7 Florida Supreme Court order, according to a recent announcement by The Florida Bar.
Boca Raton attorney disbarred following wire fraud guilty plea
TALLAHASSEE — Longtime Boca Raton attorney Jeffrey Greenfield Klein has been voluntarily disbarred following a March 14 Florida Supreme Court order after his guilty plea last year to wire fraud, according to a recent announcement by The Florida Bar.
Former Boca Raton attorney permanently disbarred following securities fraud guilty plea
Former Boca Raton attorney Mark Fisher has been voluntarily and permanently disbarred following a Feb. 7 Florida Supreme Court order after his guilty plea late last year to securities fraud, according to a recent announcement by the Florida Bar.
Imprisoned Tampa attorney disbarred over insider trading conviction
TALLAHASSEE — Tampa attorney Walter C. Little has been disbarred following an Oct. 15 Florida Supreme Court order over his securities fraud sentencing last year, according to a recent announcement by The Florida Bar.
Punta Gorda attorney permanently disbarred following 'ill-fated drug informant scheme'
Imprisoned Punta Gorda attorney Steven Paul Burch has been permanently disbarred after a heroin trafficking conviction in what the U.S. Department of Justice called an "ill-fated drug informant scheme," according to a recent announcement by the Florida Bar.
Former FSU attorney disbarred following conviction in child sex sting
TALLAHASSEE — Longtime Tallahassee attorney Dayton Michael Cramer has been disbarred following an Oct. 15 Florida Supreme Court order following his conviction earlier this year for trying to have sex with a 13-year-old girl, according to a recent announcement by The Florida Bar.
Boca Raton attorney faces voluntarily disbarment following money laundering charge
TALLAHASSEE (Florida Record) — Boca Raton attorney Lawrence Michael Weisberg faces voluntarily disbarment following a Sept. 27 Florida Supreme Court order regarding a federal money laundering charge filed against him last spring, according to a recent announcement by The Florida Bar.
Miami attorney disbarred following 2013 federal wire fraud conviction
The Florida Supreme Court disbarred Miami attorney Rafael Ubieta, who is serving a sentence for wire fraud, on Aug. 31.
Tampa attorney suspended following sentencing for insider trading
TALLAHASSEE (Florida Record) – Tampa attorney Walter C. Little has been suspended indefinitely following an April 2 Florida Supreme Court order after he was sentenced in February for insider trading, a recent announcement by The Florida Bar said.
New Jersey attorney disbarred in Florida over multiple complaints
TALLAHASSEE (Florida Record) — Miami attorney Cary G. Blake, a member of the New Jersey State Bar Association, has been disbarred following a March 8 Florida Supreme Court order regarding allegations in a multiple client matter, according to a recent announcement by The Florida Bar.
Benefits for borrowers cited from multistate PHH Mortgage settlement
This month's announced $45 million multistate settlement, which included Florida, with New Jersey-based PHH Mortgage Corporation will benefit certain borrowers who faced foreclosure and current borrowers as well, a Florida attorney general spokesman said during a recent interview.
Bentley III of Florida's middle district one of Trump's U.S. Attorney casualties
TAMPA — At the beginning of March, President Donald Trump asked for the resignation of 46 U.S. attorneys, most of whom were appointed by former president Barack Obama. One of those attorneys who turned in their resignations to the president and attorney general Jeff Sessions was A. Lee Bentley III, United States Attorney for the Middle District of Florida.
McKesson reaches $150M settlement over alleged violations
LAKELAND, Fla. — One of the largest distributors of pharmaceutical drugs in the United States has agreed to a landmark settlement, according to a report on Street Insider.
Florida woman ordered to pay more than $600K for BP oil-spill scam
MIAMI – A South Florida woman has been ordered to pay more than $600,000 in restitution for filing fraudulent claims in connection with the Deepwater Horizon explosion and oil spill in the Gulf of Mexico, a district judge recently ruled.
Attorney claims Hillsborough firefighter wanted on misdemeanor battery charge has not fled the state
TAMPA — A Hillsborough County firefighter wanted on a misdemeanor battery charge stemming from his alleged involvement in a Key West bar fight maintains he will turn himself in to face the charges, but has no intention of altering his vacation plans that would require him to return to Florida early.
The late Norman Wolfinger honored with Public Service Lifetime Achievement Award
MELBOURNE -- Norman Wolfinger, former state attorney in Brevard and Seminole counties, veteran and advocate for veterans and children, was posthumously awarded with the public service lifetime achievement award from Space Coast Daily.
Florida urged to follow DOJ's footsteps in phasing out private prisons
WASHINGTON – The U.S. Justice Department recently announced that it is ending its relationship with private prison companies, citing poor results in safety, services and affordability. The new inspector general's report found that contract facilities were inferior to government-run ones.
Sunshine state attorney seeks website changes, and costs and fees, from snow shoe seller
BOCA RATON - A small Vermont firm selling snow shoes and other winter equipment has found itself in the cross hairs of a Florida law firm demanding settlement over claims of discrimination against disabled people.


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Street Art workshop
Creating and realizing a street art painting with Tapiz Mural.
For this first painting together, we choose geometric shapes. We were quickly in phase with each others even if we were from various cultures and had different experiences. After few sessions sketching and prototyping, we went painting in the street, in Villa Crespo neighborhood.


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Having a business card today involves pride and refinement for each representative. It raises the standard of your business or the business you work for. The cards hold the personality of the individual it has a place with. In such situations, numerous business card planning organizations have ventured in to advance their cards on the web.
With the rising significance of computerized media in brand advancement, today every is trying to stamp their online nearness. Moreover, many card structuring organizations or producers are declaring their custom cards on the web. The online advancement of such custom cards despite the fact that is by all accounts simple, however the decision of a correct business configuration may not be so. An exact measure of research about various brands producing the cards may give the clients a knowledge into a relative examination of the equivalent. This near investigation may encourage the clients to pick the correct business card on the web.
What are the Essential Elements of a Business Card?
The basic considerations of any card are the name of cardholder, title, and nature of the business or association he works for, address, telephone no, email id, and site address or the business url. Moreover, the custom business cards do contain extra data. This extra data is comprehensive of occupation title of the cardholder, the slogan of the business element, business logo and the administrations rendered by the business. The previous data shapes the obligatory consideration of the business card while the later one is reliant on the individual decision.
What are the Mandatory Rules to be Considered while Designing Acustomized Business Card Online?
Coming up next is a rundown of the obligatory checkpoints to be considered while planning a business card:
Incorporate just the fundamental and genuine data: If the card speaks to the personality of the cardholder, it additionally speaks to the standard of conduct of the concerned individual. An all around organized or an efficient business card characterizes the composed working example of the person. To get an efficient card, limit the data to be incorporated on it. The more data individuals stuff in, the clumsier the shows up.
Underline the Legibility of the data: Designing a card isn't simply including pertinent data and giving it over to the clients. By intelligible, it implies that the text style utilized for including the information ought to be effectively clear to the individual getting the card. Rather than the text style utilized, the card ought to be formal, calm and expert in appearance.
Remember the focused on group of onlookers: the decision of the cards ought to be made with the end goal that it bids to the crowd at first look. The general population who handle two distinct callings in multi day ought to guarantee that they tweak singular cards for every one of their occupations.
These are a portion of the fundamental checkpoints to be considered while picking an altered card online as it speaks to the cardholder from various measurements.