- by Patel, N., Stockley, P. G.Efficient recovery and removal of nucleic acids remain major challenges in molecular biology, diagnostics, and viral-vector manufacturing. Conventional precipitation methods often require organic solvents, low temperatures, and extended centrifugation, while nuclease-based contaminant removal can be incomplete and/or is incompatible with downstream workflows. Here, we evaluate NADA, a trivalent rare earth-based reagent, for rapid, solvent-free precipitation of RNA and DNA, and ALGO-mediated recovery of the full range of properties of unmodified RNA. Using hepatitis B virus pre-genomic RNA, lambda bacteriophage DNA, […]
- by Figueiredo, A. Q., Domingos, I. F., Coelho, S., Cabral, M. d. g., Lopes, M., Carvalho, L. B., Lopez-Fernandez, H., Capelo, J. L., Lodeiro, C., Dhir, R., Jacinto, A., Wisniewski, J. R., Moroncini, G., Santos, H. M.Rationale & Objective Early prediction of renal non-recovery after acute kidney injury (AKI) could support risk-adapted follow-up and clinical decision-making. We evaluated whether a urinary fatty acid-binding protein 3 (FABP3)/apolipoprotein A-I (APOA1) ratio based on Total Protein Approach (TPA) quantification could discriminate renal recovery from non-recovery after severe AKI. Study Design Exploratory biomarker discovery study with outcome-blinded independent validation. Setting & Participants Critically ill adults with KDIGO stage 2 or 3 AKI. Urine was collected 48 hours after AKI diagnosis. […]
- by Quel Pinol, C., Chenuel, T., Henri, J.Photosynthetic carbon fixation is catalyzed by 11 enzymes in the Calvin-Benson-Bassham cycle, a highly conserved metabolic pathway in the green lineage. Fructose-1,6-bisphosphatase (FBPase) contributes to the regeneration of the substrate of carboxylation. While non-photosynthetic paralogs of FBPases contribute to cytosolic neoglucogenesis in a well-documented manner, chloroplast FBPases specific regulations by phospho- and redox-posttranslational modifications, pH, cofactor binding, and homo-oligomeric exchange is only partially understood. In particular, FBPases from green microalgae are potentially target of a multiplicity of disulfide/dithiol exchanges that […]
- by Zhang, Y., Su, D., Li, H., Wang, H., Li, J., Shi, C., Shen, T., Hou, Z., Ying, Z., Xiang, S.HP1 undergoes liquid-liquid phase separation (LLPS), yet the prevailing model, which attributes condensation almost entirely to intrinsically disordered regions (IDRs), leaves the role of its folded domains unresolved. Using solution and solid-state NMR, we show that HP1 LLPS is instead governed by reciprocal interplay between IDRs and folded domains. Within the condensed phase, solid-state NMR reveals a dynamic hierarchy: the chromoshadow domain (CSD) assembles into polymorphic oligomers that form rigid cores, while the phosphorylation sites on the N-terminal extension (NTE) […]
- Decoupling of CO2 and O2 fluxes reveals oxygen-independent decarboxylation in anoxic mammalian cellsby Vellama, H., Eichelmann, H., Gross, U., Luuk, H., Eskla, K.-L.Canonical understanding of cellular respiration holds that CO2 production tracks oxygen consumption, and mammalian cells deprived of oxygen are thought to rely on lactate fermentation, which yields no net CO2. Because standard respirometry infers CO2 flux either from oxygen consumption or from extracellular acidification rather than measuring it directly, a CO2 flux decoupled from oxygen would go undetected or be misattributed to glycolysis. Using direct measurement of CO2 and O2 fluxes from the gas phase in anoxic mammalian cells, we […]
- by Maji, D., Dingeldein, L., Li, M., Astore, M. A., Evans, L., Tang, W. S., Rogal, J., Rangan, A., Covino, R., de Marco, A., Bonilla, S. L., Cossio, P.Functional RNA structures are typically far more dynamic than most proteins solved by cryo-electron microscopy (cryo-EM). Flexible junctions and weak tertiary contacts produce broad conformational ensembles, and cryo-EM particle classification analyses discard unaligned particles, which inherently contain the information needed to quantify such broad ensembles. Here, we infer compositional and conformational distributions from unclassified particle images by Bayesian ensemble reweighting. We tested our approach on the P4-P6 domain of the Tetrahymena thermophila group I intron, a classic model system of […]
- by Mohan, I., Martin Viney, L., Batchelor, M., Richards, M. W., Ochi, T., Bayliss, R.Aurora-A kinase (AurA) is a serine/threonine kinase regulated by protein binding partners such as TPX2, which interacts with the Y-pocket of the kinase domain. Although the canonical functions of AurA are mitotic, where it phosphorylates spindle-associated proteins such as TACC3, there is evidence for non-mitotic, chromatin-associated roles. Heterogenous nuclear ribonucleoprotein K (hnRNPK) is a transcriptional regulator and an AurA substrate, how AurA is activated in this pathway is unknown. Their interaction contributes to cancer-associated nuclear accumulation of AurA and has […]
- by Olaosebikan, I. A., Courtney, K. C., Karatas Bristow, H.Transcription factors are critical regulators of homeostasis, and disruption of their function is a common underlying mechanism in cancer. The homeobox Myeloid ecotropic integration site-1 (Meis1) and its binding partner Homeobox A9 (HoxA9) are among the most highly dysregulated transcription factors in cancer, particularly in Acute Myeloid Leukemia (AML). Upregulation of HoxA9 is observed in more than 50% of AML cases and is a predictor of poor prognosis. Meis1 forms a cooperative DNA complex with HoxA9 and is required for […]
- by Kasaju, M., Palumbo, R. M., Gray, C., Grimaldi, L., Shoulders, J., Heisler, D. B., Mihailescu, M. R.Heterogenous nuclear riboprotein K (hnRNP K) is an important regulator of multiple signaling pathways involved in DNA repair and function whose expression has been found to be mis-regulated in many cancers and neurological diseases. The expression regulation of hnRNP K is not fully understood however it has been shown that the microRNA miR-1249-3p targets its mRNA 3-untranslated region (UTR), regulating its translation. In this study we identified a guanosine-rich sequence in the HNRNPK 3-UTR within the miR-1249-3p binding site that […]
- by Sahrawat, A. S., Radner, F., Gruber, K., Gruber, C., Egger, N., Zuellig, T.Adipose triglyceride lipase (ATGL) catalyses the first and rate-limiting step of intracellular triacylglycerol (TAG) hydrolysis, yielding diacylglycerol (DAG) and a free fatty acid (FA). The site of TAG cleavage determines the regioisomeric identity of the DAG product, with distinct consequences for downstream signalling and lipid metabolism. Here, we show that ATGL prefers specific fatty acyl chain lengths and that this preference contributes to its regioselectivity on heterogeneous TAG substrates. Structural modelling identified a substrate-binding pocket comprising a hydrophilic anchor and […]
- by Gheflati, H., Steiner, C., Feizy, N.Rhodopsin 1 (Rh1), the major visual pigment of Drosophila photoreceptors, requires a vitamin A-derived chromophore for proper folding, maturation and transport to the rhabdomere. While the endocytic trafficking of mature, light-activated Rh1 to endolysosomal compartments is well characterized, the fate of immature Rh1 during defective ER maturation remains unclear. Here, we developed a Rh1::eGFP-mCherry tandem reporter to monitor Rh1 localization and compartment acidification in vivo. Exploiting the acid stability of mCherry, this reporter enables visualization of Rh1 after delivery to […]
- by Peris, M., Sahuquillo, J., Ortuno, A., Pons, V., Rierola, M., Poca, M. A.Red blood cell (RBC) transfusion aims to restore tissue oxygen delivery, yet a stored unit's capacity to unload oxygen depends on 2,3-diphosphoglycerate (2,3-DPG), the principal allosteric modulator of hemoglobin-oxygen affinity. Randomized trials comparing fresher with older units have been consistently null, and all have used storage duration as the exposure variable. We asked whether storage age reflects the metabolic state of individual units. Twenty-four leukodepleted packed RBC units were sampled serially from day 3 to day 42 of standard cold […]
- by Miranda-Zaragoza, B., Gaytan, P., Huerta-Miranda, G., Ramirez-Silva, L., Miranda-Hernandez, M., Rodriguez-Almazan, C.Laccases belong to the multicopper oxidase family (MCO). Compared with fungal laccases, bacterial laccases have high stability and activity under extreme pH and temperature conditions. Because of these characteristics, bacterial laccases have become highly impactful in industrial processes. In the present work, we engineered the Thermus thermophilus HB27 laccase (Tth-Lac) by directed evolution. The library had a size of 1.2 x105 colonies, and we screened mutants by malachite green decolorization on solid medium. The results showed that Q36H mutant exhibited […]
- by Wong, J. S. K., Loke, M. S., Pradhan, B., Kanno, T., Björkegren, C., Kim, E.The Smc5/6 complex (Smc5/6) preserves genome stability through ATP-dependent DNA organization, but the structural details that prime the complex for productive DNA engagement remain unknown. Here, we combine cryo-electron microscopy and conformational heterogeneity analysis to define the pre-catalytic activation pathway of Smc5/6. We show that in the absence of ATP and DNA, Smc5/6 exists as a heterogeneous conformational ensemble composed predominantly of two conformations termed Bridge and Cross. In the presence of DNA alone, Smc5/6 adopts a Juxtaposed conformation that […]
- by Moura, A. C., Wiseman, B., Bozkurt, E., Deery, E., Pickersgill, R. W., Brindley, A. A., Hitchcock, A., Hoegbom, M., Warren, M. J., Shvarev, D.Cobalamin is a complex yet essential cobalt-containing cofactor synthesized exclusively by prokaryotes and archaea. Aerobic cobalamin biosynthesis requires the CobNST multi-subunit class I cobaltochelatase for cobalt incorporation. Cobalt chelation is dependent on the energy transfer from the CobST AAA+/VWA motor unit to the substrate-binding catalytic unit (CobN). However, the mechanism for assembly and function of CobNST cobaltochelatase remains poorly understood. Here, we used cryo-EM to solve the structures of the AAA+ motor CobS, in the absence and presence of ATP […]
- by Maita, N., Kim, S., Kajino, Y., Kato, M.TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein whose low-complexity domain (LCD) mediates self-assembly, liquid-liquid phase separation, and pathological aggregation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies have established the central ultraconserved region (UCR) of the LCD as a major determinant of TDP-43 self-association. However, the contribution of the evolutionarily conserved C-terminal region remains poorly understood. To identify LCD residues required for self-assembly, we systematically analyzed 47 ALS-associated substitutions distributed throughout the TDP-43 LCD […]
- by Deng, Q., Xu, M., Chen, H., Lei, J.Peptide offers a powerful modality for targeting protein-protein interfaces, yet their computational design remains challenging because sequence identity, backbone geometry and side-chain packing must be optimized simultaneously within a three-dimensional pocket constrained environment. Here we present PeTFlow, a pocket-conditioned all-atom peptide co-design framework that rationalizes this constrained design problem through discrete side-chain state tokenization. In PeTFlow, each residue is represented as a chemically compatible state token, enabling unified modeling of peptide sequence, conformation and all-atom interface geometry. PeTFlow achieved superior […]
- by Kroetenheerdt, E., Ehrl, A., Stelmach, C., Reina, F., Piech, L., Praetorius, F., Ries, J. A., Zagrovic, B., Leonard, T. A.Many protein kinases, ubiquitous signal transducers across all kingdoms of life, are activated by phosphorylation of one or more conserved residues in their activation loops. This reaction is widely considered to be intermolecular (trans) in nature, dependent on the transient dimerization of two kinase domains. However, since the substrate, product and reaction chemistry are identical, bulk solution kinase assays cannot distinguish between intra- and inter-molecular reactions. Discriminating between these two mechanisms is crucial because they impose different requirements on the […]
- by Makhro, A., Seiler, E., Bender, N., Eppenberger, P., Hobbs-Ray, C., Baerlocher, G.BACKGROUND: The protein 4.1a/4.1b ratio is an intrinsic molecular marker of mean red blood cell (RBC) age resulting from progressive deamidation of protein 4.1b during RBC circulation. Despite its potential for estimating mean RBC age from a single blood sample, its application has been limited by labor-intensive RBC membrane preparation, and its interindividual variability has not been characterized in a large human population. METHODS: We developed a low-volume method for direct RBC membrane isolation from whole blood using saponin permeabilization […]
- by Du, J., Zhu, S., Puja, A., McNeel, R., Alabdallat, D., Ngo, T.Purpose: The neural retina and retinal pigment epithelium/choroid (RPE/Cho) are metabolically coupled. Metabolic dysfunction of the RPE/Cho contributes to photoreceptor death in age-related macular degeneration (AMD). This study aimed to determine how aging, a major risk factor for AMD, alters the proteome and metabolic pathways in the retina and RPE/Cho. Methods: We performed quantitative proteomics on neural retina and RPE/Cho from young (8-week) and old (73-week) C57BL/6J mice. The differentially expressed proteins between old and young, as well as between […]
Mass spectrometry websites, social media, journal feeds, and other links and items of interest to the mass spectrometry community.
