- by Bate, J., Hardinge, P., Jathoul, A. P., Wilson, M. R., Murray, J. A. H.Museum collections of Coleoptera contain genetic material of potential interest to biotechnology, and non-destructive DNA extraction enables the preservation of important specimens with concomitant release of mitochondrial and genomic DNA. Mini-barcoding of regions of the mitochondrial cytochrome oxidase subunit I (MT-COI) gene helps identify and eliminate known species from further investigation. Here we identify a novel luciferase gene, using Consensus Degenerate Hybrid Oligonucleotide (CODEHOP) primers targeting the region of the luciferase gene spanning the fourth exon, intron, and fifth exon […]
- by David, M., Adam, K.-P., Li, D., Lim, X. Y., Hurrell, J. G. R., Preston, S., Peake, D. A., Batarseh, A.Lipid metabolism is increasingly recognized as a hallmark of cancer, yet translating lipidomic discoveries into clinically actionable biomarkers remains constrained by analytical variability and limited standardized validation frameworks. This challenge is further compounded by a chicken-or-egg problem, where expensive standards and labelled internal standards are required to identify and quantitate target lipids, but the diagnostic importance of these targets is uncertain until they can be reliably measured. Previous work had indicated the potential of 48 lipid biomarker species for the […]
- by Paidi, S. K., Ibrahim, J., Stepurska, K., Zarzar, J., Izadi, S., Rude, E., Luu, S., Kovner, D., O'Connor, K., Bol, K., Mehta, S., Andersen, N., Stephens, N., Makowski, E., Heisler, J., Swartz, T., Carter, P. J., Baginski, T.Predicting high-concentration viscosity of monoclonal antibodies such as IgG1 is crucial for their development as therapeutics for subcutaneous delivery. Unfortunately, traditional experimental rheometry methods for assessing viscosity are low-throughput. This study evaluates Self-Interaction Nanoparticle Spectroscopy (SINS) assays – specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS) – for high-throughput viscosity prediction. We characterized 96 IgG1 antibodies, assessing SINS against in silico descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, […]
- by Sarrain, E. J., Wang, Q., Bondoy, A. C., Guo, F., Cao, Q., Niu, H.Ribonucleotides are frequently incorporated into our genome during replication. Canonically, RNase H2 is responsible for the removal of these embedded ribonucleotides. Alternatively, DNA topoisomerase 1 (TOP1) has also been shown to have genomic ribonucleotide processing activity. When this process occurs at short tandem repeat (STR) sequences, it can lead to 2-5 bp deletions. These deletions are the result of two sequential cuts by TOP1 at sites of ribonucleotide incorporation. In this study, we have determined that PARP1 regulates the TOP1-mediated […]
- by Hau, K., Fecke, A., Hormann, F.-L., Groba, A.-C., Melo, L. M. N., Cansiz, F., Allies, G., Hentschel, A., Chen, J., Heiles, S., Tasdogan, A., Sickmann, A., Smith, K. W.Technological advances in biomedical sciences have accelerated multi-omics research, enabling high-resolution spatial mapping of diverse molecular compound classes. However, integrating spatial omics often requires serial tissue sections, limiting the alignment correlation across modalities. We present a single-section integrative multi-omics (SIMO) workflow that combines metabolite and lipid imaging with histopathology and region-specific proteomics. Using MALDI-MSI, tissue staining, and laser microdissection (LMD), SIMO delivers comprehensive metabolic, lipidomic, and proteomic insight from the same sample. Using mouse cardiac tissue we develop, control, and […]
- by Karpauskaite, L., Voisin, T. B., Maslen, S. L., Kent, L., Pellowe, G. A., Skehel, M., Balchin, D.Protein folding is fast relative to mRNA translation and nascent polypeptides begin to fold during their synthesis on the ribosome. The resulting cotranslational folding intermediates are accessible to diverse molecular chaperones that recognise incompletely folded client proteins. Here, we sought to understand how specific chaperone:client complexes are established during protein synthesis, using the chaperone-dependent tumor suppressor protein p53 as a model. By capturing interactomes at different points during p53 synthesis, we show that Hsp70, Hsp90 and TRiC bind competitively to […]
- by Dhakal, A., Gyawali, R., Cheng, J.Cryo-electron tomography (cryo-ET) enables in situ three-dimensional visualization of many protein complexes and other macromolecular assemblies such as ribosomes in cells, yet automated macromolecule particle identification in 3D cryo- ET tomograms remains a major bottleneck due to dose-limited low signal-to-noise ratios, missing-wedge artifacts, and densely crowded cellular backgrounds. We present TomoSwin3D, an end-to-end three-dimensional (3D) macromolecule particle identification and classification pipeline centered on a Swin Transformer-based U-Net that performs particle identification and classification and outputs particle centroid coordinates. TomoSwin3D leverages […]
- by Thalhammer, K. O., Scurria, M., Li, J., Trindade, I. B., Gutierrez, O., Conway, S. J., Newman, D. K.Phenazines are redox-active microbial metabolites produced and secreted in diverse ecological contexts from soils to chronic infections. In these disparate environments phenazines can function variously as antibiotics, extracellular electron shuttles, and nutrient scavengers. Key to understanding the impact of these functions is a robust expectation of phenazine retention or diffusion in a given context. But predicting phenazine fate and transport is difficult because of the chemical complexity of their local microenvironments. To address this challenge, we measured the octanol water […]
- by Khanppnavar, B., Florea, R., Schuster, D., Lavriha, P., Kosturanova, A., Ruckstuhl, M. M., Kantarci, I., Vaithia, A., Shi, J., Picotti, P., Gossert, A. D., Leitner, A., Korkhov, V. M.Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal […]
- by Donaghy, C. M., Heyer-Gray, H., O'Hern, C., Ibrahim, M., Perez-Torres, B., Bogues, N., Alexandrescu, A., Djoko, K., Klassen, J. L., Angeles-Boza, A. M.Host defense peptides (HDPs) are important components of the innate immune system that are used to combat pathogens and often rely on metal binding for their function. However, controlling trace nutrients such as transition metals may have other roles in host-symbiont interactions beyond poisoning harmful pathogens. This study characterizes the evolution, structural properties, and biochemical activity of the novel hymenopteran HDP abaecin-2. In myrmicine ants such as the fungus-growing tribe Attini, abaecin-2 has evolved to include an Amino-Terminal Cu(II) and […]
- by Germer, P., Gericke, L., Koeppl, L.-H., Zou, Z., Jockmann, E., Kuge, M., Zoller, K., Herrmann, H., Fuderer, R., Mohr, M. K. F., Bartels, A., Oral, G., Lukat, P., Layer, G., Mueller, M., Blankenfeldt, W., Barra, L., Andexer, J. N.The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine instead of the native cofactor S-adenosyl-L-methionine remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the […]
- by Nokhoijav, E., Kaplar, M., Aranyi, S. C., Berzi, A., Bergström, G., Antonopoulos, K., Edfors, F., Emri, M., Csosz, E.Obesity and Type 2 Diabetes (T2D) are heterogeneous metabolic disorders whose molecular diversity is incompletely defined. We analyzed circulating proteomic profiles from 129 individuals belonging to Control, Obesity, and T2D groups and applied complementary machine-learning approaches, including random forest, multinomial logistic regression with LASSO regularization, support vector machines, and ensemble voting to identify proteins distinguishing the clinical groups. Convergent model outputs revealed a partially overlapping panel of discriminative proteins. Model performance was evaluated in an independent dataset from the Human […]
- by Rumyantseva, A., Wilhalm, A., Carter, W., Tippetts, T. S., Moedas, M., Rosenberger, F. A., Moore, D., Vegvari, A., Hinze, Y., Muellner-Wong, L., Alsina, D., Wibom, R., Winston, R., Mathews, T. P., Lund, L. H., Andersson, D. C., Pironti, G., Wedell, A., DeBerardinis, R. J., Freyer, C., Wredenberg, A.The neonatal heart undergoes a rapid metabolic transition from fetal glycolysis to oxidative phosphorylation, requiring coordinated metabolic remodeling. Mechanisms driving this transition remain unclear. Here, we demonstrate that sufficient mitochondrial S-adenosylmethionine (mitoSAM), imported via the solute carrier Slc25a26, is essential for this shift by sustaining the lipoylation of 2-oxoacid dehydrogenases, critical for TCA cycle activation. Proteomic and metabolomic profiling revealed that reduced mitoSAM availability impaired lipoylation, blocking TCA cycle function and restricting nucleotide synthesis, while mitochondrial gene expression and respiratory […]
- by Di Geronimo, B., Zuson, J., Udzenija, A., Chanique, A., Kourist, R., Kamerlin, S. C. L.Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN […]
- by Kantarci, I., Haoriwa, H., Korkhov, V. M.Adenylyl cyclases (ACs) convert ATP into the second messenger cAMP, thus directly influencing cellular signaling in response to a wide variety of stimuli. Despite their physiological importance, structural studies of isoform-specific AC regulation are compounded by difficulties in AC expression and purification. Here, we designed a chimeric construct AC95, combining human AC9 as a molecular scaffold and incorporating the catalytic-allosteric core of human AC5. Cryo-EM analysis of AC95 at 3.5 [A] resolution revealed a state of AC95 bound to both […]
- by Shi, Y., Liu, X., Chen, L.Nitric oxide (NO) signaling is pivotal in numerous physiological processes and is implicated in a spectrum of human diseases. Nitric oxide synthases (NOS) initiate NO signaling and govern its magnitude and duration, making them key drug targets. Despite decades of investigation, the structural mechanism by which NOS enzymes transfer electrons from NADPH to haem remains incompletely understood. Here, we report cryo-electron microscopy studies of the inducible NOS (iNOS) homodimer in complex with calmodulin captured under the catalytic turnover condition, resolving […]
- by Jaber, Q. Z., Yu, N. J., Masuda, I., Hou, Y.-M., Kleiner, R. E.RNA modifications play an important role in biological processes. Mapping the diversity of RNA chemistry and studying the biological function of individual modifications remains an outstanding challenge in many organisms. In particular, RNA modifications remain poorly studied across most bacterial systems. Our group previously developed RNA-mediated activity-based protein profiling (RNABPP), a reactivity-based strategy employing metabolic labeling and quantitative proteomics to profile RNA modification writer enzymes in human cells. Here we adapt this approach to characterize RNA-modifying enzymes in bacteria. We […]
- by Messa, P. E., Warren, C. L., Nicol, N. R., Pearson, K. S., Peters, J. P., Fowler, A. M., Alarid, E. T., Ozers, M. S.Grainyhead-like 2 (GRHL2) is an epithelial transcription factor with context-dependent regulatory roles, yet the sequence rules governing its DNA recognition remain incompletely defined. In this study, a high-density genomic Specificity and Affinity for Protein (SNAP) DNA-binding array containing 772,732 tiled probes derived from GRHL2 ChIP-seq regions was used to resolve GRHL2 binding specificity at 6 base pair resolution across genomic sequences. From high-affinity probes, de novo motif analysis recovered the canonical 5-AACCGGTT-3 motif. Sequence specificity landscapes revealed a stepwise reduction […]
- by Wu, J., Chen, X., Zhou, K., Wang, W.Atherosclerosis (AS) is a chronic inflammatory disease closely linked to vascular senescence, yet the specific molecular mechanisms connecting aging processes to AS pathogenesis remain incompletely understood. This study integrated transcriptomic data from GEO datasets (GSE100927 and GSE43292) to identify vascular aging-related differentially expressed genes (VARDEGs). Following batch effect correction, 28 VARDEGs were screened and subjected to functional enrichment, protein-protein interaction (PPI) network analysis, and immune infiltration assessment. Seven hub genes (MMP9, APOE, TNF, ICAM1, PPARG, CYBA, and NCF2) were identified […]
- by Richter, S. M., Bui, H.-L., Chen, A., Tannous, C., Butler, B. R., Bennett, S. D., Nguyen, S. Q.-a., Prado, J., Mohamed, A., DuBois, I. A., Tadros, E., Thai, N. T., Lima Guan, S., Peralta, C. M., Kwong, A., Hawk, L. M. L., Grazioli, G., Wang, N.The NAD+ dependent deacetylase sirtuin-1 (SIRT1) is known to elicit cellular defenses against aging, cancer, and other aberrant pathologies. Previous studies have identified an intrinsically disordered region of SIRT1 comprised of N-terminal residues 1-52, herein referred to as motif A, which activates SIRT1 activity, likely through intramolecular interactions. Additionally, phosphorylation of N-terminal residues Ser27 and Ser47 has been shown to be important for regulating SIRT1 activity and stability. The lack of in vitro characterization of these effects hampers our further […]
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