Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.
The Lim domain only 2 (Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3–31 and 66–105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human β-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.
Background: Obstructive sleep apnea syndrome (OSAS) occurs more frequently in Alzheimers disease (AD) than in controls. OSAS is characterized by the obstruction of the upper airway resulting in nocturnal intermittent hypoxia. Hypoxia has been found to be related to the Alzheimer biomarkers amyloid-beta and tau in animal studies. The aim of the present study was to investigate whether a history of OSAS is associated with cerebrospinal fluid (CSF) levels of amyloid-beta 42 (AB42), total tau (tau) and phosphorylated tau (ptau) in patients with subjective cognitive decline (SCD), mild cognitive impairment (MCI), and AD. Methods: We included 59 patients who reported OSAS in their medical history (age 63 (8) years, 11 (19%) female; 14 AD, 15 MCI, 30 SCD) from the Amsterdam Dementia Cohort. We matched these patients for baseline diagnosis, gender, age, and year of evaluation, on a 1:2 basis to patients without an OSAS history (N¼118). CSF AB42, tau and ptau were assessed using ELISA (Innotest). We assessed the association between OSAS and the CSF biomarkers with Linear Mixed Models (LMM), that included terms for OSAS, diagnosis, and the interactions between OSAS and diagnosis. All models were adjusted for APOE E4 status. Results:In the total sample there was no association between OSAS and CSF biomarkers. The relationship between OSAS and tau tended to be different between diagnostic groups (p-value interaction .095). Post-hoc analysis showed that AD patients with a history of OSAS had higher tau levels than those without OSAS (estimated mean [95% CI]: 845 vs. 647 [549-744] pg/ml, p-value ¼ .02). OSAS was not associated with tau in SCD and MCI patients. There was no interaction between OSAS and diagnosis for CSF AB42 and ptau. Conclusions: We found that a history of OSAS is associated with higher CSF tau levels in AD. These findings could fit with the idea that hypoxia might have an additive effect to neuronal injury in AD, or vice versa, that patients with high tau levels are at increased risk of OSAS. However, inferences on causal relationships cannot be made given the observational nature of the study.Background: Alzheimer's disease (AD) is one of the most common neurodegenerative diseases of the world. One of its major hallmarks is the hyperphosphorylation of tau protein leading to intracellular neurofibrillary tangles (NFTs) in neurons. These tangles represent an end stage of paired helical filament (PHF) aggregation, which are found in neuropil threads in early stages of dementia. The hyperphosphorylation occurs at different tau protein residues like serine, threonine and tyrosine, including several phospho-sites especially related to AD. However, appearance, quantity and location of NFTs are still not fully understood. Methods: Using indirect immunofluorescence and quantitative image analysis, tau pathologies in diseased and healthy human brain, as well as transgenic and non-transgenic APP SL mice were investigated. We analyzed three different phospho-sites (pSer199, pSer396 and pSer422) in cortical regions and ...
mitocondrial membrane potential, ATP prodction, and cell viability. Cellular ROS level was significantly decreased after treatment of KST013418. Therapeutic effects of KST013418 were demonstrated from the in vivo assays, as well. Conclusions: KST013418 and its derivatives can be used to activate the neuropretective mechanism by recovering mitochondrial dysfunction. This development of novel TSPO ligands supported that the discovery of potent TSPO ligands must be a meaningful way for curing AD patients.Background: The pathological hallmarks of AD is characterized by clumps of aggregated beta-amyloid (Ab) ("plaques") and tau ("tangles"), which along with their monomeric forms are thought to be pathologically benign. Sporadic or templated misfolding of these monomeric proteins initiates an oligomerization process to generate intermediate transient oligomeric species (OAb and Otau) that display a multitude of neurotoxic and synaptotoxic effects. We have identified a novel druggable target known as the Common Conformational Motiff (CCM) which is shared on both Ab and tau. We have identified a novel, drug like, CNS penetrable class of compounds capable of binding to the CCM and preventing oligomerization of both Ab and tau. Methods: Efficacy of the TRV 200 class in preventing oligomerization was measured in vitrousing biotin-beta-amyloid and biotin-tau assays. The compounds were screened in several cell-based tau aggregation assays and analysed by multiple biophysical techniques to monitor protein-ligand interactions. ADMET and DMPK was collected on numerous compounds within the series. Results: TRV 200 class demonstrated prevention of oligomerization activity against both beta-amyloid and tau. Compounds from this class showed attenuation of tau misfolding as studied by mass spec and electrochemical techniques. Lead compounds from the TRV 200 class are selective, demonstrating favourable in vitro ADMET and DMPK with appropriate CNS penetration. Conclusions: We have designed and developed a new class of compounds capable of inhibiting oligomerization of both beta-amyloid and tau proteins. Our small molecules have appropriate pharmacokinetic profiles and are able to reach the target tissue and engage and attenuate both targets in several in vivo models of AD.
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