Chemical Probe Design & Development for Assessing Protein Modification and Cross-Linking

Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the capacity of antioxidants in a cell. The CNS is particularly sensitive to oxidative stress and increased oxidative stress is implicated in the development and progression of neurodegenerative diseases. One of the results of increased oxidative stress is the peroxidation of polyunsaturated fatty acids (PUFAs). Lipid peroxides then undergo degradation and produce electrophilic lipid peroxidation products (LPx) which include 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE). These LPx contain two reactive functionalities that result in the formation of proteins adducts through different mechanisms. The aldehyde is capable of reacting with the terminal amino group of Lys residues to produce reversible Schiff base protein adducts. Additionally, the reaction of Cys, His, or Lys residues with the α,β-unsaturated aldehyde results in the formation of an irreversible Michael addition adduct. This bivalent reactivity has been shown to promote the formation of protein oligomers, implicated in neurodegenerative diseases, such as Aβ, tau, and α-synuclein. The methods commonly used to detect these protein adducts are chemical derivatization, immunoblotting, or LC-MS/MS analysis. These methods suffer from different limitations, therefore, we have developed a dual color near-IR imaging method that allows for the visualization and quantitation of the Schiff base adducts, Michael addition adducts, and protein crosslinking induced by these LPx. By adapting this method we were also able to enrich these modified proteins and use LC-MS/MS to identify HNE protein adducts in the lysates of neuronal SHSY-5Y cells. Several nucleophilic small molecules have been reported to protect tissues and cells from conditions of increased oxidative stress by behaving as chemical traps of LPx. It has also been reported that the trapping of LPx by these small molecules results in a decreased level of LPx protein adduct formation. We have shown that these trapping agents react with LPx in vitro and protect against the death of SHSY-5Y cells induced by the treatment of exogenous LPx. Our imaging method demonstrates that trapping agents containing a free thiol (glutathione, N-acetyl-cysteine) significantly decrease the formation of protein adducts. However, this method shows that the neuroprotective activity observed from certain aldehyde reactive scavengers (histidyl-hydrazide, hydralazine) is a caused by a prevention of protein cross-linking, not an overall decrease in protein adduct formation

Interaction of oxidative stress and neurotrauma in ALDH2−/− mice causes significant and persistent behavioral and pro-inflammatory effects in a tractable model of mild traumatic brain injury

Oxidative stress induced by lipid peroxidation products (LPP) accompanies aging and has been hypothesized to exacerbate the secondary cascade in traumatic brain injury (TBI). Increased oxidative stress is a contributor to loss of neural reserve that defines the ability to maintain healthy cognitive function despite the accumulation of neuropathology. ALDH2-/- mice are unable to clear aldehyde LPP by mitochondrial aldehyde dehydrogenase-2 (Aldh2) detoxification and provide a model to study mild TBI (mTBI), therapeutic interventions, and underlying mechanisms. The ALDH2-/- mouse model presents with elevated LPP-mediated protein modification, lowered levels of PSD-95, PGC1-α, and SOD-1, and mild cognitive deficits from 4 months of age. LPP scavengers are neuroprotective in vitro and in ALDH2-/- mice restore cognitive performance. A single-hit, closed skull mTBI failed to elicit significant effects in WT mice; however, ALDH2-/- mice showed a significant inflammatory cytokine surge in the ipsilateral hemisphere 24 h post-mTBI, and increased GFAP cleavage, a biomarker for TBI. Known neuroprotective agents, were able to reverse the effects of mTBI. This new preclinical model of mTBI, incorporating significant perturbations in behavior, inflammation, and clinically relevant biomarkers, allows mechanistic study of the interaction of LPP and neurotrauma in loss of neural reserve

American Gut Project fecal sOTU counts table

The Deblur sOTU counts table for the fecal samples used in the American Gut Project manuscript. The samples were trimmed to a common read length of 125nt, and processed by Deblur (Amir et al mSystems 2017). Blooms were removed (Amir et al mSystems 2017) and any sample with fewer than 1250 sequences was omitted. This table is not rarefied,

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Placing changes in the microbiome in the context of the American Gut. We accumulated samples over sequencing runs to demonstrate the structural consistency in the data. We demonstrate that while the ICU dataset (https://www.ncbi.nlm.nih.gov/pubmed/27602409) falls within the American Gut samples, they do not fall close to most samples at any of the body sites. We then highlight samples from the United Kingdom, Australia, the United States and other countries to show that nationality does not overcome the variation in body site. We then highlight the utility of the American Gut in meta-analysis by reproducing results from (https://www.ncbi.nlm.nih.gov/pubmed/20668239) and (https://www.ncbi.nlm.nih.gov/pubmed/23861384), using the AGP dataset as the context for dynamic microbiome changes instead of the HMP dataset. We show rapid, complete recovery of C. diff patients following fecal material transplantation and also contextualized the change in an infant gut over time until it settles into an adult state. This demonstrates the power of the American Gut dataset, both as a cohesive study and as a context for other investigations

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The SEPP (Mirarab et al Pac Symp Biocomput 2012) fragment insertion tree used for phylogenetic analyses

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Longitudinal samples from a large bowel resection. We place longitudinal samples collected prior to and following a large bowel resection in the context of samples from the AGP, the Earth Microbiome Project (https://www.ncbi.nlm.nih.gov/pubmed/29088705), intensive care unit patients (https://www.ncbi.nlm.nih.gov/pubmed/27602409), "extreme" diet samples from (https://www.ncbi.nlm.nih.gov/pubmed/24336217), and samples from the Hadza hunter-gatherers (https://www.ncbi.nlm.nih.gov/pubmed/28839072). Unweighted UniFrac was computed on this sample set, and principal coordinates were assessed. Using EMPeror (https://www.ncbi.nlm.nih.gov/pubmed/24280061), we then animate the plot by connect successive data points gut resection time series, while rotating the data frame. We first show the how the extent of change in the microbial community, and how the samples immediately following surgery resemble fecal samples from ICU patients. In the background of the animation, a black line connects a plant rhizosphere sample to a marine sediment sample, which have the same unweighted UniFrac distance (0.78) as the longitudinal sample immediately preceding and immediately following surgery

Unweighted UniFrac distances

The unweighted UniFrac distance (Lozupone and Knight AEM 2005) matrix of the 9511 fecal samples used in the American Gut paper. UniFrac was computed using Striped UniFrac (https://github.com/biocore/unifrac). Prior to execution of UniFrac, Deblur (Amir et al mSystems 2017) was run on the samples, all bloom sOTUs were removed (Amir et al mSystems 2017), and samples were rarefied to a depth of 1250 reads (Weiss et al Microbiome 2017). For the phylogeny, fragments were inserted using SEPP (Mirarab et al Pac Symp Biocomput 2012) into the Greengenes 13_5 99% OTU tree (McDonald et al ISME 2012)

Full American Gut Project mapping file

The full American Gut Project mapping file, includes non-fecal samples

Weighted normalized UniFrac distances

The weighted normalized UniFrac distance (Lozupone et al AEM 2007) matrix of the 9511 fecal samples used in the American Gut paper. UniFrac was computed using Striped UniFrac (https://github.com/biocore/unifrac). Prior to execution of UniFrac, Deblur (Amir et al mSystems 2017) was run on the samples, all bloom sOTUs were removed (Amir et al mSystems 2017), and samples were rarefied to a depth of 1250 reads (Weiss et al Microbiome 2017). For the phylogeny, fragments were inserted using SEPP (Mirarab et al Pac Symp Biocomput 2012) into the Greengenes 13_5 99% OTU tree (McDonald et al ISME 2012)

American Gut Project fecal sOTU relative abundance table

The Deblur sOTU relative abundance table for the fecal samples used in the American Gut Project manuscript. The samples were trimmed to a common read length of 125nt, and processed by Deblur (Amir et al mSystems 2017). Blooms were removed (Amir et al mSystems 2017) and any sample with fewer than 1250 sequences was omitted. This table is not rarefied, and is normalized to 1