bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB
Authors: Acharya Ananya , Bret Hélène , Huang Jen-Wei , Mütze Martin , Göse Martin , Kissling Vera , Seidel Ralf , Ciccia Alberto , Guérois Raphaël , Cejka Petr 
Published: June 2023

AbstractThe human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.
Understanding the Influence of Context on Real-World Walking Energetics
Authors: Baroudi Loubna , Barton Kira , Cain Stephen M. , Shorter K. Alex 
Published: June 2023

ABSTRACTSpeeds that minimize energetic cost during steady-state walking have been observed during lab-based investigations of walking biomechanics and energetics. However, in real-world scenarios, humans walk in various contexts that elicit different walking strategies, which may not always prioritize minimizing energetic cost. To investigate whether individuals tend to select energetically optimal speeds in real-world situations and how contextual factors influence gait, we conducted a study combining data from lab and real-world experiments. Walking kinematics and context were measured during daily life over a week (N = 17) using wearable sensors and a mobile phone. To determine context, we utilized self-reported activity logs, GPS data, and follow-up exit interviews. Additionally, we estimated energetic cost using respirometry over a range of gait speeds in the lab. Cost of transport during these trials was used to identify an energetically optimal walking speed range for each participant. The proportion of real world steady-state stride speeds within this range was identified for all data and for each context. We found that participants walked significantly faster than what was energetically optimal, and the proportion of steady-state strides within the energetically optimal speed range was dependent on the context. On average, 45.6% of all steady-state stride speeds were energetically optimal across all contexts for all participants. These results suggest that while energetic cost is a factor considered by humans when selecting gait speed in daily life, it is not the sole determining factor. Context contributes to the observed variability in movement parameters both within and between individuals.
Impact ofBmal1rescue and time-restricted feeding on liver and muscle proteomes during the active phase in mice
Authors: Smith Jacob G. , Molendijk Jeffrey , Blazev Ronnie , Chen Wan Hsi , Zhang Qing , Litwin Christopher , Greco Carolina M. , Sassone-Corsi Paolo , Muñoz-Cánoves Pura , Parker Benjamin L. , Koronowski Kevin B. 
Published: June 2023

ABSTRACTObjectiveMolecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding is well defined at the transcriptional level, their impact on governing protein abundances in peripheral tissues is unclear. Here, we determine the relative contributions of the local molecular clock and daily feeding cycles on liver and muscle proteomes during feeding.MethodsLC-MS/MS was performed on liver and skeletal muscle harvested four hours into the dark phase from wild-type (WT),Bmal1knockout (KO), and liver- and muscle-Bmal1-rescued (LMRE) mice housed under 12-h light/12-h dark cycles with eitherad libitumfeeding or nighttime-restricted feeding. Additional molecular and metabolic analyses were performed on liver and cultured hepatocytes.ResultsFeeding-fasting cycles had only minimal effects on liver and none on muscle. In contrast,Bmal1KO altered the abundance of 674 proteins in liver, and 80 in muscle. Rescue of liver and muscleBmal1restored 50% of proteins in liver and 25% in muscle. These included proteins involved in carbohydrate metabolism in muscle and in fatty acid oxidation in liver. For liver, proteins involved inde novolipogenesis were largely dependent onBmal1function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 were enriched for secreted proteins; we determined that the maintenance of FGF1 abundance requires liver BMAL1, and that autocrine signaling through FGF1 is necessary and sufficient for mitochondrial respiration in hepatocytes.ConclusionsBMAL1 in liver and muscle is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and numerous metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.
A nucleoid-associated protein is involved in the emergence of antibiotic resistance by promoting the frequent exchange of the replicative DNA polymerase inM. smegmatis
Authors: Ng Wei L. , Rego E. Hesper 
Published: June 2023

SUMMARYAntibiotic resistance inM. tuberculosisexclusively originates from chromosomal mutations, either during normal DNA replication or under stress, when the expression of error-prone DNA polymerases increases to repair damaged DNA. To bypass DNA lesions and catalyze error-prone DNA synthesis, translesion polymerases must be able to access the DNA, temporarily replacing the high-fidelity replicative polymerase. The mechanisms that govern polymerase exchange are not well understood, especially in mycobacteria. Here, using a suite of quantitative fluorescence imaging techniques, we discover that, as in other bacterial species, inM. smegmatis,the replicative polymerase, DnaE1, exchanges at a timescale much faster than that of DNA replication. Interestingly, this fast exchange rate depends on an actinobacteria-specific nucleoid-associated protein (NAP), Lsr2. In cells missinglsr2, DnaE1 exchanges less frequently, and the chromosome is replicated more faithfully. Additionally, in conditions that damage DNA, cells lackinglsr2load the complex needed to bypass DNA lesions less effectively and, consistently, replicate with higher fidelity but exhibit growth defects. Together, our results show that Lsr2 promotes dynamic flexibility of the mycobacterial replisome, which is critical for robust cell growth and lesion repair in conditions that damage DNA.ImportanceUnlike many other pathogens,M. tuberculosishas limited ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. Thus, the mechanisms that facilitate chromosomal mutagenesis are of particular importance in mycobacteria. Here, we show that Lsr2, a nucleoid-associated protein, has a novel role in DNA replication and mutagenesis in the model mycobacteriumM. smegmatis. We find that Lsr2 promotes the fast exchange rate of the replicative DNA polymerase, DnaE1, at the replication fork and is important for the effective loading of the DnaE2-ImuA’-ImuB translesion complex. Withoutlsr2,M. smegmatisreplicates its chromosome more faithfully and acquires resistance to rifampin at a lower rate, but at the cost of impaired survival to DNA damaging agents. Together, our work establishes Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.
refMLST: Reference-based Multilocus Sequence Typing Enables Universal Bacterial Typing
Authors: Khdhiri Mondher , Thomas Ella , de Smet Chanel , Chandar Priyanka , Madasu Vivek , Chandrakumar Induja , Davidson Jean M , Anderson Paul , Chorlton Samuel D 
Published: June 2023

AbstractSummaryCommonly used approaches for genomic investigation of bacterial outbreaks, including SNP and gene-by-gene approaches, are limited by the requirement for curated allele schemes. As a result, they only work on a select subset of known organisms, and fail on novel or less studied pathogens. We introduce refMLST, a gene-by-gene approach using the reference genome of a bacterium to form a scalable, reproducible and robust method to perform outbreak investigation. When applied to 1263Salmonella entericagenomes, refMLST enabled consistent clustering, improved resolution and faster processing in comparison to chewieSnake. refMLST is applicable to any bacterial species with a public genome, does not require a curated scheme, and automatically accounts for genetic recombination.Availability and ImplementationrefMLST is freely available for academic use athttps://bugseq.com/academic.
Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction
Authors: Hernandez Belinda J. , Skiba Nikolai P. , Plößl Karolina , Strain Madison , Grigsby Daniel , Kelly Una , Cady Martha A. , Manocha Vikram , Maminishkis Arvydas , Watkins TeddiJo , Miller Sheldon S. , Ashley-Koch Allison , Stamer W. Daniel , Weber Bernhard H.F. , Rickman Catherine Bowes , Klingeborn Mikael 
Published: June 2023

AbstractThe retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of exosomes that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral exosomes from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of exosome release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in exosome content, including basal-side specific desmosome and hemidesmosome shedding via exosomes. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases, (e.g., AMD) and broadly from blood-CNS barriers in other neurodegenerative diseases.
Characterisation ofPlasmodium vivaxlactate dehydrogenase dynamics inP. vivaxinfections
Authors: Cao Pengxing , Kho Steven , Grigg Matthew J. , Barber Bridget E. , Piera Kim A. , William Timothy , Poespoprodjo Jeanne R. , Jang Ihn Kyung , Simpson Julie A. , McCaw James M. , Anstey Nicholas M. , McCarthy James S. , Britton Sumudu 
Published: June 2023

AbstractPlasmodium vivaxlactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway ofPlasmodium vivax. It can also be used as a diagnostic biomarker. Quantitation of plasma PvLDH has been used as a measure ofP. vivaxbiomass in clinical studies of uncomplicated and severe vivax malaria. With the increasing importance of PvLDH in studyingP. vivaxdiagnosis and infection, improved characterisation of the dynamics of this biomarker is important. In this study, we developed mathematical models that capture parasite and matrix PvLDH dynamics inex vivoculture and the human host. We estimated the biological parameters usingex vivoandin vivolongitudinal data of parasitemia and PvLDH concentration collected fromP. vivax-infected humans using Bayesian hierarchical inference. We found that theex vivoandin vivoestimates of PvLDH in a parasitized red blood cell differed significantly across the asexual life cycle, within vivoestimates at least ten-fold higher thanex vivoestimates (for example, the median estimate of intraerythrocytic PvLDH mass at the end of the life cycle was 9.4×10−3ngin vivovs. 5.1×10−4ngex vivo). We also estimated theex vivoPvLDH half-life to be 65.3 h (95% credible interval: 60.8—70.7 h), which is approximately three times longer than the median estimate of thein vivoPvLDH half-life, 21.9 h (16.7—29.9 h). Our findings provide an important foundation to further improve quantitative understanding ofP. vivaxbiology and facilitate the development of PvLDH-based diagnostic tools.
Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex withClostridium perfringensenterotoxin
Authors: Erramilli Satchal K. , Dominik Pawel K. , Ogbu Chinemerem P. , Kossiakoff Anthony A. , Vecchio Alex J. 
Published: June 2023

AbstractClaudins are a family of ∼25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex withClostridium perfringensenterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.
Microglial P2Y6calcium signaling promotes phagocytosis and shapes neuroimmune responses in epileptogenesis
Authors: Umpierre Anthony D. , Li Bohan , Ayasoufi Katayoun , Zhao Shunyi , Xie Manling , Thyen Grace , Hur Benjamin , Zheng Jiaying , Liang Yue , Wu Zhaofa , Yu Xinzhu , Sung Jaeyun , Johnson Aaron J. , Li Yulong , Wu Long-Jun 
Published: June 2023

SUMMARYMicroglial calcium signaling is rare in a baseline state but shows strong engagement during early epilepsy development. The mechanism and purpose behind microglial calcium signaling is not known. By developing anin vivoUDP fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP signals to the microglial P2Y6receptor for broad increases in calcium signaling during epileptogenesis. UDP-P2Y6signaling is necessary for lysosome upregulation across limbic brain regions and enhances production of pro-inflammatory cytokines—TNFα and IL-1β. Failures in lysosome upregulation, observed in P2Y6KO mice, can also be phenocopied by attenuating microglial calcium signaling in Calcium Extruder (“CalEx”) mice. In the hippocampus, only microglia with P2Y6expression can perform full neuronal engulfment, which substantially reduces CA3 neuron survival and impairs cognition. Our results demonstrate that calcium activity, driven by UDP-P2Y6signaling, is a signature of phagocytic and pro-inflammatory function in microglia during epileptogenesis.
Circulating Sex-specific Markers of Plaque Instability in Women and Men with Severe Carotid Atherosclerosis
Authors: Gasbarrino Karina , Zheng Huaien , Daskalopoulou Stella S. 
Published: June 2023

AbstractBackgroundDifferences in plaque composition and instability exist among men and women. Circulating markers that reflect sex-specific features in the plaque should be explored for better prediction of high-risk plaques in women and in men. This study aims to 1) investigate differences in the lipid, immune, and adipokine circulating profiles of men and women with stable versus unstable plaques, and 2) identify circulating markers that can better classify men and women according to plaque instability.MethodsPre-operative blood samples as well as plaque specimens were collected from men and women undergoing a carotid endarterectomy (n=460). Blood samples were used for adipokine, lipid, and immune profiling. Plaque stability was determined by gold-standard histological classifications.ResultsMen had more unstable plaques than women (P<0.001), exhibiting greater plaque hemorrhage, a larger lipid core, and more inflammation (P<0.001), as well as less favourable circulating profiles. Significant antagonistic interactions were observed between sex and white blood cell (WBC) counts, sex and basophil to WBC ratio, and sex and platelet counts on impacting plaque instability. Several circulating immune parameters served as independent sex-specific markers of plaque instability; low total white blood cell (WBC) counts, high monocyte to WBC ratio, and low basophil to WBC ratio were associated with greater plaque instability in men, while a higher basophil to WBC ratio was observed in women with unstable plaques.ConclusionsOur findings demonstrated sex-specific differences between older men and postmenopausal women with severe carotid atherosclerosis, with women displaying more stable plaque phenotypes, and favourable circulating profiles compared to men. We identified several potential circulating markers that relate to sex-specific plaque phenotypes for better prediction of high-risk plaques in women and in men. Following future validation, these markers could be implemented into clinical practice to monitor when the plaque becomes unstable and better select men and women for intervention.
Rictor induces AKT signaling to regulate lymphatic valve formation
Authors: Banerjee Richa , Knauer Luz A. , Iyer Drishya , Barlow Sara E. , Scallan Joshua P. , Yang Ying 
Published: June 2023

AbstractLymphatic valves are specialized structures of the collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress (OSS) from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves throughout life. Conventionally, in other tissue types, AKT activation requires dual kinase activity and the mammalian target of rapamycin complex 2 (mTORC2) commands this process by phosphorylating AKT at Ser473. Here we showed that embryonic and postnatal lymphatic deletion ofRictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels.RICTORknockdown in human lymphatic endothelial cells (hdLECs) not only significantly reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions, but also abolished the upregulation of AKT activity and valve-forming genes in response to flow. We further showed that the AKT target, FOXO1, a repressor of lymphatic valve formation, had increased nuclear activity inRictorknockout mesenteric LECs,in vivo. Deletion ofFoxo1inRictorknockout mice restored the number of valves to control levels in both mesenteric and ear lymphatics. Our work revealed a novel role of RICTOR signaling in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately allows the formation and maintenance of a normal lymphatic valve.
Quantitative Analysis of Macrophage Uptake and Retention of Fluorescent Organosilica Nanoparticles
Authors: Chou Hung-Chang , Chiu Shih-Jiuan , Hu Teh-Min 
Published: June 2023

AbstractThis study investigates the uptake and retention of stable fluorescent organosilica nanoparticles by macrophages, which play a vital role in scavenging environmental nanoparticles and nanomedicine within the body. We used rhodamine 6G-loaded fluorescent organosilica nanoparticles (SiNP-R6G) synthesized from a thiol-functionalized organosilane precursor. Our primary objective was to establish a quantitative relationship between fluorescent measurements and nanoparticle tracking analysis, enabling the precise “counting” of nanoparticles taken up by macrophages under kinetic measurement conditions. Our kinetic study demonstrated a concentration-dependent, saturable internalization of nanoparticles in a model macrophage (RAW 264.7 cells), with a maximum uptake rate (Vmax) of 7.9 × 104nanoparticles per hour per cell. The estimated number concentration of nanoparticles for half-maximum uptake was approximately 0.8 trillion nanoparticles per milliliter, and a significant portion (∼80%) of internalized SiNP-R6G remained entrapped within the cells for 48 hours, indicating the sustained particle retention capacity of macrophages. These findings highlight the successful development of a methodology to accurately “count” the cellular uptake of nanoparticles in macrophages, providing valuable insights into the kinetics and retention capabilities of macrophages for nanoparticles.
From Hot Water to Dry Dirt: Microbes Use Cytochrome ‘Nanowires’ of Similar Conductivity but Different Structure
Authors: Guberman-Pfeffer Matthew J. 
Published: June 2023

AbstractMicron-scale electron transfer through polymeric cytochrome ‘nanowires’ powers prokaryotic life from hydrothermal vents to terrestrial soils in ways not fully understood. How much structural diversity optimizes electrical conductivity for survival in these different habitats is challenging to assess experimentally. Herein, physiologically relevant redox conduction is computationally assessed in cytochrome filaments fromGeobacter sulfurreducens(OmcE, OmcS, and OmcZ),Pyrobaculum calidifontis(A3MW92), andArchaeoglobus veneficus(F2KMU8). A newly implemented Python program, BioDC, is used and validated against redox currents predicted from considerably more expensive molecular dynamics and quantum mechanical/molecular mechanical calculations. BioDC uses the heme solvent accessibility, stacking geometry, and redox-linked change in electrostatic energy to estimate electron transfer energetics. Leveraging this efficiency, structurally diverse cytochrome ‘nanowires’ from different organisms are shown to have similar redox conductivities. A functionally robust heme chain ‘packaged’ in habitat-customized proteins is proposed to be a general evolutionary design principle for cytochrome ‘nanowires’ widely distributed among prokaryotes.TOC Graphic