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1. Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

   https://doi.org/10.1186/s40168-022-01454-1  

   Kleiner, Manuel ; Kouris, Angela ; Violette, Marlene ; D’Angelo, Grace ; Liu, Yihua ; Korenek, Abigail ; Tolić, Nikola ; Sachsenberg, Timo ; McCalder, Janine ; Lipton, Mary S. ; et al ( February 2023 , Microbiome)

   Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput.

   Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using¹⁸O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.

   We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

    

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2. ADEPT: a domain independent sequence alignment strategy for gpu architectures

   https://doi.org/10.1186/s12859-020-03720-1  

   Awan, Muaaz G. ; Deslippe, Jack ; Buluc, Aydin ; Selvitopi, Oguz ; Hofmeyr, Steven ; Oliker, Leonid ; Yelick, Katherine ( December 2020 , BMC Bioinformatics)

   Abstract Background Bioinformatic workflows frequently make use of automated genome assembly and protein clustering tools. At the core of most of these tools, a significant portion of execution time is spent in determining optimal local alignment between two sequences. This task is performed with the Smith-Waterman algorithm, which is a dynamic programming based method. With the advent of modern sequencing technologies and increasing size of both genome and protein databases, a need for faster Smith-Waterman implementations has emerged. Multiple SIMD strategies for the Smith-Waterman algorithm are available for CPUs. However, with the move of HPC facilities towards accelerator based architectures, a need for an efficient GPU accelerated strategy has emerged. Existing GPU based strategies have either been optimized for a specific type of characters (Nucleotides or Amino Acids) or for only a handful of application use-cases. Results In this paper, we present ADEPT, a new sequence alignment strategy for GPU architectures that is domain independent, supporting alignment of sequences from both genomes and proteins. Our proposed strategy uses GPU specific optimizations that do not rely on the nature of sequence. We demonstrate the feasibility of this strategy by implementing the Smith-Waterman algorithm and comparing it to similar CPU strategies as well as the fastest known GPU methods for each domain. ADEPT’s driver enables it to scale across multiple GPUs and allows easy integration into software pipelines which utilize large scale computational systems. We have shown that the ADEPT based Smith-Waterman algorithm demonstrates a peak performance of 360 GCUPS and 497 GCUPs for protein based and DNA based datasets respectively on a single GPU node (8 GPUs) of the Cori Supercomputer. Overall ADEPT shows 10x faster performance in a node-to-node comparison against a corresponding SIMD CPU implementation. Conclusions ADEPT demonstrates a performance that is either comparable or better than existing GPU strategies. We demonstrated the efficacy of ADEPT in supporting existing bionformatics software pipelines by integrating ADEPT in MetaHipMer a high-performance denovo metagenome assembler and PASTIS a high-performance protein similarity graph construction pipeline. Our results show 10% and 30% boost of performance in MetaHipMer and PASTIS respectively. 

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3. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

   https://doi.org/10.3390/cryst11080874  

   Oestreicher, Zachery ; Valverde-Tercedor, Carmen ; Mumper, Eric ; Pérez-Guzmán, Lumarie ; Casillas-Ituarte, Nadia N. ; Jimenez-Lopez, Concepcion ; Bazylinski, Dennis A. ; Lower, Steven K. ; Lower, Brian H. ( August 2021 , Crystals)

   null (Ed.)

   Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals. 

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4. Particle Size Specific Magnetic Properties Across the Norwegian‐Greenland Seas: Insights Into the Influence of Sediment Source and Texture on Bulk Magnetic Records

   https://doi.org/10.1029/2018GC007894  

   Hatfield, Robert G. ; Wheeler, Benjamin H. ; Reilly, Brendan T. ; Stoner, Joseph S. ; Housen, Bernard A. ( February 2019 , Geochemistry, Geophysics, Geosystems)

   We make fundamental observations of the particle size variability of magnetic properties from 71 core tops that span the southern Greenland and Norwegian Seas. These data provide the first detailed regional characterization of how bulk magnetic properties vary with sediment texture, sediment source, and sediment transport. Magnetic susceptibility (MS) and hysteresis parameters were measured on the bulk sediment and the five constituent sediment particle size fractions (clay, fine silt, medium silt, coarse silt, and sand). The median MS value of the medium silt size fraction is ~3–5 times higher than that of the sand and clay size fractions and results in a strong sensitivity of bulk MS to sediment texture. Hysteresis properties of the clay size fraction are relatively homogeneous and contrast that silt and sand size fractions which show regional differences across the study area. These coarser fractions are more transport limited and using medium silt hysteresis measurements and low temperature MS behavior we establish three endmembers that effectively explain the variability observed across the region. We model the response of bulk magnetic properties to changes in sediment texture and suggest that variations in sediment source are required to explain the bulk magnetic property variability observed in cores across the southern Greenland and Norwegian Seas. These findings imply that sediment source has a greater influence on driving bulk magnetic property variability across this region than has previously been assumed.

    

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5. Quantification of N 6 ‐formylated lysine in bacterial protein digests using liquid chromatography/tandem mass spectrometry despite spontaneous formation and matrix effects

   https://doi.org/10.1002/rcm.9019  

   Folz, Jacob S. ; Patterson, Jenelle A. ; Hanson, Andrew D. ; Fiehn, Oliver ( January 2021 , Rapid Communications in Mass Spectrometry)

   N⁶‐Formyl lysine is a well‐known modification of histones and other proteins. It can also be formed as a damaged product from direct formylation of free lysine and accompanied by other lysine derivatives such as acetylated or methylated forms. In relation to the activity of cellular repair enzymes in protein turnover and to lysine metabolism, it is important to accurately quantify the overall ratio of modified lysine to free lysine.

   N⁶‐Formyl lysine was quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data collected in a non‐targeted manner using positive mode electrospray ionization on a Q‐Exactive HF⁺Orbitrap mass spectrometer. Studies were performed with lysine and deuterated lysine spiked into protein digests and solvents to investigate the extent of spontaneous formation and matrix effects of formation ofN⁶‐formyl lysine.

   We show thatN⁶‐formyl lysine,N²‐formyl lysine,N⁶‐acetyl lysine, andN²‐acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples.N⁶‐Formyl lysine was spontaneously formed and correlated to the concentration of lysine. In the sample matrix of protein digests, 0.03% of lysine was spontaneously converted intoN⁶‐formyl lysine, and 0.005% of lysine was converted intoN⁶‐formyl lysine in pure run solvent.

   Spontaneous formation ofN⁶‐formyl lysine,N⁶‐acetyl lysine,N²‐formyl lysine, andN²‐acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples.

    

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