bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploring the effect of mechanical anisotropy of protein structures in the unfoldase mechanism of AAA+ molecular machines
Authors: Varikoti Rohith Anand , Fonseka Hewafonsekage Yasan Y. , Kelly Maria S. , Javidi Alex , Damre Mangesh , Mullen Sarah , Nugent Jimmie L. , Gonzales Christopher M. , Stan George , Dima Ruxandra I. 
Published: April 2022

AbstractEssential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of μm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. By considering wild-type and variants of DHFR, we found that optimal ClpY-mediated action probes favorable orientations of the substrate relative to the machine. Unfolding of wild-type DHFR involves strong mechanical interfaces near each terminal and occurs along branched pathways, whereas unfolding of DHFR variants involves softer mechanical interfaces and occurs through single pathways, but translocation hindrance can arise from internal mechanical resistance. For spastin, optimum severing action initiated by pulling on a tubulin subunit is achieved through the orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.
Mechanical morphotype switching as an adaptive response in mycobacteria
Authors: Eskandarian Haig Alexander , Chen Yu-Xiang , Toniolo Chiara , Belardinelli Juan M. , Palcekova Zuzana , Ashby Paul , Fantner Georg E. , Jackson Mary , McKinney John D. , Javid Babak 
Published: April 2022

AbstractInvading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which mycobacteria alter the mechanical stiffness of their cell surface to become refractory to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of “mechanical morphotype switching” in mycobacteria exposed to host intracellular stress. A “soft” mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Genetic manipulation, by deletion of uvrA, or pharmacological treatment, with bedaquiline, locked mycobacteria into a “soft” mechanical morphotype state, enhancing survival in macrophages. Our study proposes microbial mechanical adaptation as a new axis for surviving host-mediated stressors.One-Sentence SummaryBacteria alter their cell surface mechanical properties to increase survival during macrophage infection.
Characterizing a New Fluorescent Protein for Low Limit of Detection Sensing in the Cell-Free System
Authors: Copeland Caroline E. , Kim Jeehye , Copeland Pearce L. , Heitmeier Chloe J. , Kwon Yong-Chan 
Published: April 2022

ABSTRACTCell-free protein synthesis-based biosensors have been developed as highly accurate, low- cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor’s dynamic range must be increased in the system to achieve the low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein - mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder Green Fluorescent Protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce false positives proportion.
A Lipid Atlas of the Human Kidney
Authors: Farrow Melissa A. , Tideman Léonore E.M. , Neumann Elizabeth K. , Patterson Nathan Heath , Migas Lukasz G. , Colley Madeline E. , Allen Jamie L. , Rivera Emilio S. , Romer Carrie E. , Yang Haichun , Brewer Maya , Pingry Ellie , Dufresne Martin , Djambazova Katerina , Sharman Kavya , Kruse Angela R.S. , Gutierrez Danielle B. , Harris Raymond C. , Fogo Agnes B. , de Caestecker Mark P. , Caprioli Richard M. , Van de Plas Raf , Spraggins Jeffrey M. 
Published: April 2022

ABSTRACTTissue atlases provide foundational knowledge on the cellular organization and molecular distributions across molecular classes and spatial scales. Here, we construct a comprehensive spatio-molecular lipid atlas of the human kidney from 29 donor tissues using integrated multimodal molecular imaging. Our approach leverages high spatial resolution matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) for untargeted lipid mapping, stained microscopy for histopathological assessment, and tissue segmentation using autofluorescence microscopy. With a combination of unsupervised, supervised, and interpretive machine learning, the atlas provides multivariate lipid profiles of specific multicellular functional tissue units (FTUs) of the nephron, including the glomerulus, proximal tubules, thick ascending limb, distal tubules, and collecting ducts. In total, the atlas consists of tens of thousands of FTUs and millions of mass spectrometry measurements. Detailed patient, clinical, and histopathologic information allowed molecular data to be mined based on these features. As examples, we highlight the discovery of how lipid profiles are altered with sex and differences in body mass index.
Neuronopathic GBA1 L444P mutation accelerates Glucosylsphingosine levels and formation of hippocampal alpha-synuclein inclusions
Authors: Mahoney-Crane Casey L. , Viswanathan Megha , Russell Dreson , Curtiss Rachel A.C. , Freire Jennifer , Bobba Sai Sumedha , Coyle Sean D. , Kandebo Monika , Yao Lihang , Wan Bang-Lin , Hatcher Nathan G. , Smith Sean M. , Marcus Jacob N. , Volpicelli-Daley Laura A. 
Published: April 2022

ABSTRACTThe most common genetic risk factor for Parkinson’s disease (PD) is heterozygous mutations in the GBA1 gene which encodes for the lysosomal enzyme, glucocerebrosidase (GCase). GCase impairments are associated with an accumulation of abnormal α-synuclein (α-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the GBA1 L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444Pmice to determine the effects of this severe GBA1 mutation on lipid metabolism, expression of synaptic proteins, behavior, and α-syn inclusion formation. GBA1+/L444Pmice showed reduced GCase activity in limbic brain regions and expressed lower levels of hippocampal vGLUT1 compared to wildtype (GBA1+/+) mice. GBA+/L444Pmice also demonstrated impaired fear conditioning, but no motor deficits. We show, using mass spectrometry, that mutant GCase and age increased levels of glucosylsphingosine (GlcSph), but not glucosylceramide (GlcCer), in the brains and serum of GBA1+/L444Pmice. Aged GBA1+/+mice also showed increased levels of GlcSph, and decreased GlcCer. To model disease pathology, templated α-syn pathology was used. α-Syn inclusions were increased in the hippocampus of GBA1+/L444Pmice compared to GBA1+/+mice, but not in the cortex, or substantia nigra pars compacta (SNc). Pathologic α-syn did not cause a loss of dopamine neurons in the SNc. Treatment with a GlcCer synthase inhibitor prevented loss of cortical α-syn inclusions, but not loss of dopamine neurons. Overall, these data suggest the critical importance to evaluate the contribution of hippocampal pathologic α-syn and brain and serum glucosylsphingosine in synucleinopathies.SIGNIFICANCE STATEMENTSynucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB), are both pathologically characterized by abnormal α-synuclein (α-syn). Mutant GBA1 is a risk factor for both PD and DLB where a reduction of glucocerebrosidase (GCase) activity is seen. Collectively, this indicates the significance of evaluating mutant GCase in synucleinopathies. Our data suggest the critical importance to evaluate the contribution of hippocampal pathologic α-syn and brain and serum glucosylsphingosine (GlcSph) accumulation in synucleinopathies. Moreover, these pathologic outcomes may contribute to the nonmotor symptoms clinically observed in PD and DLB. Our findings highlight the importance of GlcSph as a relevant biomarker for future therapeutics.
Antibiotic persistence of Brucella abortus in its protective intracellular niche
Authors: Mode Selma , Ketterer Maren , Québatte Maxime , Dehio Christoph 
Published: April 2022

AbstractBackgroundHuman Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections associated with high morbidity. Treatment typically involves a combination therapy of two antibiotics administered for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon.Methodology/Principal findingsWe used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular niche to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments.Conclusion/SignificanceWe demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche. Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation.Authors SummaryBrucellosis is a zoonosis endemic to many low- and middle-income countries around the world. Recommended therapies by the WHO are comprised of at least two antibiotics for several weeks, sometimes month. Despite these harsh treatments relapses are frequent. The underlying reasons for these relapses, besides reinfection and non-compliance to treatment, are unknown. Our study shows that Brucella abortus can form so called “persisters” under recommended treatments in rich broth but also inside macrophages. This small bacterial subpopulation survives antibiotic treatment and resumes growth after removal of the antibiotics and could therefore serve as a reservoir for relapses in human Brucellosis. Furthermore, we show that the macrophage intracellular niche of Brucella has protective properties against recommended antibiotics as observed for other intracellular pathogens, highlighting the necessity to develop new infection models to assess antibiotic efficacy.
The Statistical Trends of Protein Evolution: A Lesson from AlphaFold Database
Authors: Tang Qian-Yuan , Ren Weitong , Wang Jun , Kaneko Kunihiko 
Published: April 2022

AbstractThe recent development of artificial intelligence provides us with new and powerful tools for studying the mysterious relationship between organism evolution and protein evolution. In this work, based on the AlphaFold Protein Structure Database (AlphaFold DB), we perform comparative analyses of the proteins of different organisms. The statistics of AlphaFold-predicted structures show that, for organisms with higher complexity, their constituent proteins will have larger radii of gyration, higher coil fractions, and slower vibrations, statistically. By conducting normal mode analysis and scaling analyses, we demonstrate that higher organismal complexity correlates with lower fractal dimensions in both the structure and dynamics of the constituent proteins, suggesting that higher functional specialization is associated with higher organismal complexity. We also uncover the topology and sequence bases of these correlations. As the organismal complexity increases, the residue contact networks of the constituent proteins will be more assortative, and these proteins will have a higher degree of hydrophilic-hydrophobic segregation in the sequences. Furthermore, by comparing the statistical structural proximity across the proteomes with the phylogenetic tree of homologous proteins, we show that, statistical structural proximity across the proteomes may indirectly reflect the phylogenetic proximity, indicating a statistical trend of protein evolution in parallel with organism evolution. This study provides new insights into how the diversity in the functionality of proteins increases and how the dimensionality of the manifold of protein dynamics reduces during evolution, contributing to the understanding of the origin and evolution of lives.
Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS
Authors: Guo Naici , Minas Giorgos , Synowsky Silvia A. , Dunne Margaret R. , Ahmed Hasnain , McShane Rhiannon , Bhardwaj Anshul , Donlon Noel E. , Lorton Cliona , O’Sullivan Jacintha , Reynolds John V. , Caie Peter D. , Shirran Sally L. , Lynch Andy G. , Stewart Alan J. , Arya Swati 
Published: April 2022

AbstractOesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3vsTRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, GSTP1 was found to exhibit the lowest p-value and highest classification accuracy and Cohen’s kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers.
Chemical conversion of human conventional Pluripotent Stem Cells to Trophoblast Stem Cells
Authors: Zorzan Irene , Betto Riccardo Massimiliano , Rossignoli Giada , Arboit Mattia , Drusin Andrea , Martini Paolo , Martello Graziano 
Published: April 2022

AbstractIn human embryos, naive pluripotent cells of the inner cell mass generate epiblast, primitive endoderm and Trophectoderm (TE) lineage, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and can generate trophoblast stem cells (TSCs), while conventional PSCs form amnion-like cells and lack the competence to generate TSCs. Transient histone deacetylase and MEK inhibitions with LIF stimulation can be used to chemically reset conventional to naive PSCs. Here we report that chemical resetting induced expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allowed for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a responsive intermediate state, in which conventional PSCs rapidly acquire competence to form TSCs without the need of stabilisation and expansion in a naive state. The efficiency and rapidity of our system will be useful for the study of cell fate transitions, and to generate models of placental disorders.
Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex
Authors: Pinotsis Nikos , Krüger Anna , Tomas Nicolas , Chatziefthymiou Spyros D , Litz Claudia , Mortensen Simon Arnold , Daffé Mamadou , Marrakchi Hedia , Antranikian Garabed , Wilmanns Matthias 
Published: April 2022

ABSTRACTThe identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge onin vivorelevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.
A consensus view on the folding mechanism of protein G, L and their mutants
Authors: Chang Liwei , Perez Alberto 
Published: April 2022

ABSTRACTMuch of our understanding of folding mechanisms comes from interpretations of experimental ϕ and ψ value analysis – relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of protein G, L and their mutants. Protein G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the sameprioron all systems and correctly identifies the folding mechanism for each of the four proteins – a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with Markov State Model provide a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follows the order of events observed in the folding pathways.
The NLRP3 inflammasome selectively drives IL-1β secretion byPseudomonas aeruginosainfected neutrophils and regulates bacterial killingin vivo
Authors: Minns Martin S. , Liboro Karl , Lima Tatiane S. , Abbondante Serena , Miller Brandon A. , Marshall Michaela E. , Tran-Chau Jolynn , Rietsch Arne , Dubyak George R. , Pearlman Eric 
Published: April 2022

ABSTRACTMacrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1β secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected withPseudomonas aeruginosastrain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) stimulated NLRC4 for IL-1β secretion. While IL-1β release fromΔexoSTinfected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model ofP. aeruginosacorneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 inP. aeruginosakeratitis.