bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular mechanism of Mad2 conformational conversion promoted by the Mad2-interaction motif of Cdc20
Authors: Yu Conny W.H. , Fischer Elyse S. , Greener Joe G. , Yang Jing , Zhang Ziguo , Freund Stefan M.V. , Barford David 
Published: March 2024

AbstractDuring mitosis, unattached kinetochores trigger the spindle assembly checkpoint by promoting assembly of the mitotic checkpoint complex, a heterotetramer comprising Mad2, Cdc20, BubR1 and Bub3. Critical to this process is the kinetochore-mediated catalysis of an intrinsically slow conformational conversion of Mad2 from an open (O-Mad2) inactive state to a closed (C-Mad2) active state bound to Cdc20. These Mad2 conformational changes involve substantial remodelling of the N-terminal β1 strand and C-terminal β7/β8 hairpin.In vitro, the Mad2- interaction motif (MIM) of Cdc20 (Cdc20MIM) triggers rapid conversion of O- to C-Mad2, effectively removing the kinetic barrier for MCC assembly. How Cdc20MIMdirectly induces Mad2 conversion remains unclear. In this study we demonstrate that the Cdc20MIM-binding site is inaccessible in O-Mad2. Time-resolved NMR and molecular dynamics simulations show how Mad2 conversion involves sequential conformational changes of flexible structural elements in O-Mad2, orchestrated by Cdc20MIM. Conversion is initiated by the β7/β8 hairpin of O-Mad2 transiently unfolding to expose a nascent Cdc20MIM-binding site. Engagement of Cdc20MIMto this site promotes release of the β1 strand. We propose that initial conformational changes of the β7/β8 hairpin allows binding of Cdc20MIMto a transient intermediate state of Mad2, thereby lowering the kinetic barrier to Mad2 conversion.
Cellular mechanotransduction of human osteoblasts in microgravity
Authors: Wubshet Nadab H. , Cai Grace , Chen Samuel J. , Sullivan Molly , Reeves Mark , Mays David , Harrison Morgan , Varnado Paul , Yang Benjamin , Arreguin-Martinez Esmeralda , Qu Yunjia , Lin Shan-Shan , Duran Pamela , Aguilar Carlos , Giza Shelby , Clements Twyman , Liu Allen P. 
Published: March 2024

AbstractAstronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.
The p90 Ribosomal S6 Kinases 2 and 4 promote Prostate Cancer cell proliferation in androgen-dependent and independent ways
Authors: Cronin Ryan , Azadova Aygun , Marco Antonio , Laissue Philippe P. , Brooke Greg N. , Prischi Filippo 
Published: March 2024

AbstractOncogenic activation of the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways are frequent events in Prostate Cancer (PCa) that have been correlated to tumour formation, disease progression and therapeutic resistance. At the intersection of these two pathways lies the p90 ribosomal S6 kinase (RSKs) family, which regulates many proteins involved in cell survival, growth and proliferation. As such, deregulated RSKs activity has been associated with multiple cancer types, including PCa. However, the full extent of the RSKs involvement in prostate tumorigenesis remains to be determined. Here we have shown that RSKs levels are increased in PCa samples and cell lines. The RSKs were found to enhance Androgen Receptor (AR) activity, the key oncogenic driver in PCa. Indeed, all RSKs were found to interact in close proximity to the AR. However, RSK2/4, but not RSK1/3, showed changes in cell localisation following AR nuclear translocation. Consistently, silencing of RSK2/4, but not RSK1/3, inhibited PCa proliferation in an androgen-dependent and independent manner, respectively, and induced different signaling events downstream of the AR. The data suggests that RSK2 and RSK4 activity is required for PCa cell proliferation, but they are likely regulating growth via different mechanisms.
In Vitro Budding and Floatation based Enrichment of Mitochondria-derived Vesicles for Proteomics from Rat Heart
Authors: Nair Nidhi , Ramesh Lariza , Marib Areeba , Rajeev Thejaswitha , Johnson John B , Sundararaman Ananthalakshmy 
Published: March 2024

SummaryMitochondria-derived vesicles (MDVs) are a novel class of vesicles whose biogenesis and trafficking are poorly characterized. Here, we describe the protocol toin vitrogenerate MDVs from mitochondria isolated from animal heart tissues. The protocol can be coupled with Transmission Electron Microscopy (TEM), proteomics or western blotting as final readouts. The protocol is optimized to improve the yield and purity of vesicles for downstream proteomics and facilitates selection of key regulators to be validated within cells.Graphical abstractThe protocol has been adapted and further modified from (Soubannier et al., 2012).
Axonal Injury, Sleep Disturbances, and Memory following Traumatic Brain Injury
Authors: Tinney Emma M. , España-Irla Goretti , Warren Aaron E.L. , Whitehurst Lauren N. , Stillman Alexandra M , Hillman Charles H. , Morris Timothy P. ,   Alzheimer’s Disease Neuroimaging Initiative
Published: March 2024

AbstractObjectivesTraumatic brain injury (TBI) is associated with sleep deficits, but it is not clear why some report sleep disturbances and others do not. The objective of this study was to assess the associations between axonal injury, sleep, and memory in chronic and acute TBI.MethodsData were acquired from two independent datasets which included 156 older adult veterans (69.8 years) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) with prior moderate-severe TBIs and 90 (69.2 years) without a TBI and 374 participants (39.6 years) from Transforming Research and Clinical Knowledge in TBI (TRACK-TBI) with a recent mild TBI (mTBI) and 87 controls (39.6 years), all who completed an MRI, memory assessment, and sleep questionnaire.ResultsOlder adults with a prior TBI had a significant association between axial diffusivity in the left anterior internal capsule (ALIC) and sleep disturbances [95% CI(5.0e+07, 1.7e+08), p ≤ .01]. This association was significantly different [95% CI(6.8e+07,2.2e+08), p=.01] from controls. ALIC predicted changes in memory over one-year in TBI [95% CI(−1.8e+08,-2.7e+07), p=.03]. We externally validated those findings in TRACK-TBI where ALIC axial diffusivity within two-weeks after injury was significantly associated with higher sleep disturbances in the TBI group at two-weeks [95% CI(−7.2e-06, −1.9e-04), p=0.04], six-months [95% CI(−4.2e-06,-1.3e-04), p≤ .01] and 12-months post-injury [95% CI(−5.2e-06, −1.2e-04), p=0.03]. These associations not seen in controls.InterpretationsAxonal injury to the ALIC is robustly associated with sleep disturbances in multiple TBI populations. Early assessment of ALIC damage following mTBI could identify those at risk for persistent sleep disturbances following injury.
Sorting at ciliary base and ciliary entry of BBSome, IFT-B and IFT-A
Authors: Mitra Aniruddha , Gioukakis Evangelos , Mul Wouter , Peterman Erwin J.G. 
Published: March 2024

Summary/AbstractAnterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging inC. eleganschemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results indicate that the assembly of IFT trains is a step-wise process involving the subsequent incorporation of first IFT-B, then IFT-A and finally BBSomes.
Overlapping binding sites underlie TF genomic occupancy
Authors: Khetan Shubham , Bulyk Martha L. 
Published: March 2024

AbstractSequence-specific DNA binding by transcription factors (TFs) is a crucial step in gene regulation. However, current high-throughputin vitroapproaches cannot reliably detect lower affinity TF-DNA interactions, which play key roles in gene regulation. Here, we developed PADIT-seq (proteinaffinity toDNA byin vitrotranscription and RNAsequencing) to assay TF binding preferences to all 10-bp DNA sequences at far greater sensitivity than prior approaches. The expanded catalogs of low affinity DNA binding sites for the human TFs HOXD13 and EGR1 revealed that nucleotides flanking high affinity DNA binding sites create overlapping lower affinity sites that together modulate TF genomic occupancyin vivo. Formation of such extended recognition sequences stems from an inherent property of TF binding sites to interweave each other and expands the genomic sequence space for identifying noncoding variants that directly alter TF binding.One-Sentence SummaryOverlapping DNA binding sites underlie TF genomic occupancy through their inherent propensity to interweave each other.
Contact behaviour before, during and after the COVID-19 pandemic in the Netherlands: evidence from contact surveys in 2016-2017 and 2020-2023
Authors: Backer Jantien A. , Vos Eric R. A. , den Hartog Gerco , van Hagen Cheyenne C. E. , de Melker Hester E. , van der Klis Fiona R. M. , Wallinga Jacco 
Published: March 2024

AbstractBackgroundThe first wave of the COVID-19 pandemic in 2020 was largely mitigated by reducing contacts in the general population. In 2022 most contact-reducing measures were lifted.AimWe assess whether the population has reverted to pre-pandemic contact behaviour and how this would affect the transmission potential of a newly emerging pathogen.MethodsThe PIENTER Corona study was held every 2-6 months in the Netherlands from April 2020, as a follow-up on the 2016-2017 PIENTER3 study. In both studies, participants (ages 1-85) reported the number and age group of all face-to-face persons contacted on the previous day. The contact behaviour during and after the COVID-19 pandemic was compared to the pre-pandemic baseline. The transmission potential was examined using the Next Generation Matrix approach.ResultsWe found an average of 15.4 (14.3-16.4, 95% CI) community contacts per person per day in the post-pandemic period, which is 13% lower than the baseline value of 17.8 (17.0-18.5). Children have the highest number of contacts as before the pandemic. Mainly adults aged 20-59 have not reverted to their pre-pandemic behaviour, possibly because this age group works more often from home. Although the number of contacts is structurally lower compared to the pre-pandemic period, the effect on the transmission potential of a newly emerging respiratory pathogen is limited if all age groups were equally susceptible.ConclusionContinuous monitoring of contacts can signal changes in contact patterns and can provide a ‘new normal’ baseline. Both aspects are needed to be prepared for a future pandemic.
Up-regulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain
Authors: Agostini Francesco , Pereyra Leonardo , Dale Justin , Yambire King Faisal , Maglioni Silvia , Schiavi Alfonso , Ventura Natascia , Milosevic Ira , Raimundo Nuno 
Published: March 2024

ABSTRACTMitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in the cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Nevertheless, the signaling consequences of primary mitochondrial malfunction and of primary lysosomal defects are not similar, despite in both cases there are impairments of mitochondria and of lysosomes. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects, to identify what are the global cellular consequences that are associated with malfunction of mitochondria or lysosomes. We used these data to determine what are the pathways that are affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. This pathway is transcriptionally up-regulated in cellular and mouse models of lysosomal defects and is transcriptionally down-regulated in cellular and mouse models of mitochondrial defects. We identified a role for post-transcriptional regulation of the transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, the retention of Ca2+in the lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verifiedin vivo, using models of mitochondria-associated diseases in C.elegans, that normalization of lysosomal Ca2+levels results in partial rescue of the developmental arrest induced by the respiratory chain deficiency.
MYT1L deficiency impairs excitatory neuron trajectory during cortical development
Authors: Yen Allen , Chen Xuhua , Skinner Dominic D. , Leti Fatjon , Crosby MariaLynn , Hoisington-Lopez Jessica , Wu Yizhe , Chen Jiayang , Mitra Robi D. , Dougherty Joseph D. 
Published: March 2024

AbstractMutations that reduce the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. Furthermore, MYT1L is routinely used as a proneural factor in fibroblast-to-neuron transdifferentiation. MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss. In this study, we profiled 313,335 nuclei from the forebrains of wild-type and MYT1L-deficient mice at two developmental stages: E14 at the peak of neurogenesis and P21, when neurogenesis is complete, to examine the role of MYT1L levels in the trajectory of neuronal development. We found that MYT1L deficiency significantly disrupted the relative proportion of cortical excitatory neurons at E14 and P21. Significant changes in gene expression were largely concentrated in excitatory neurons, suggesting that transcriptional effects of MYT1L deficiency are largely due to disruption of neuronal maturation programs. Most effects on gene expression were cell autonomous and persistent through development. In addition, while MYT1L can both activate and repress gene expression, the repressive effects were most sensitive to haploinsufficiency, and thus more likely mediate MYT1L syndrome. These findings illuminate the intricate role of MYT1L in orchestrating gene expression dynamics during neuronal development, providing insights into the molecular underpinnings of MYT1L syndrome.
Comparing the limbic-frontal connectome across the primate order: conservation of connections and implications for translational neuroscience
Authors: Folloni Davide , Roumazeilles Lea , Bryant Katherine L , Manger Paul R , Bertelsen Mads F , Khrapitchev Alexandre A , Rudebeck Peter H , Mars Rogier B 
Published: March 2024

ABSTRACTThe interaction of the limbic system and frontal cortex of the primate brain is important in many affective behaviors. For this reason, it is heavily implicated in a number of psychiatric conditions. This system is often studied in the macaque monkey, the most largely-used non-human primate model species. However, how evolutionary conserved this system is and how well results obtained in any model species translate to the human can only be understood by studying its organization across the primate order. Here, we present an investigation of the topology of limbic-frontal connections across seven species, representing all major branches of the primate family tree. We show that dichotomous organization of amydalofugal and uncinate connections with frontal cortex is conserved across all species. Subgenual connectivity of the cingulum bundle, however, seems less prominent in prosimian and New World monkey brains. These results inform both translational neuroscience and primate brain evolution.
Adaptation of a methanogen to the constructed environment
Authors: Kawaichi Satoshi , Kotoky Rhitu , Fiutowski Jacek , Rotaru Amelia-Elena 
Published: March 2024

AbstractDue to unique genomic adaptations, Methanococcus maripaludis Mic1c10 exhibits severe corrosive behavior when in direct contact with Fe0. These adaptations are linked to attachment and effective growth on constructed surfaces. One such adaptation is that of a specific [NiFe]-hydrogenase that may anchor on the cell surface via glycosyl-glycosyl interactions to receive Fe0-electrons directly. Such an evolutionary response to constructed environments requires us to rethink methane cycling in human-altered ecosystems.
Nano-scale viscometry reveals an inherent mucus defect in cystic fibrosis
Authors: Ponomarchuk Olga , Boudreault Francis , Gryczynski Ignacy , Dzyuba Sergei V. , Fudala Rafal , Gryczynski Zygmunt , Hanrahan John W. , Grygorczyk Ryszard 
Published: March 2024

AbstractAbnormally viscous and thick mucus is a hallmark of cystic fibrosis (CF). How the genetic defect causes abnormal mucus in CF remains unanswered and a question of paramount interest. Mucus is produced by hydration of gel-forming mucin macromolecules that are stored in secretory granules prior to release. Current understanding of mucin/mucus structure before and after secretion remains limited and contradictory models exist. Here we used a molecular viscometer and fluorescence lifetime imaging of primary epithelial cells (Normal and CF) to measure nanometer-scale viscosity. We found significantly elevated intraluminal nanoviscosity in a population of CF mucin granules, indicating an intrinsic, pre-secretory, mucin defect. Validation experiments showed that high nanoviscosity in cellular environments is mainly due to the low mobility of water that hydrates macromolecules. Nanoviscosity influences protein conformational dynamics and function. Its elevation along the protein secretory pathway indicates molecular overcrowding and is expected to alter mucin’s post-translational processing, hydration, and mucus rheology after release. The nanoviscosity of extracellular CF mucus was elevated compared to non-CF mucus. Remarkably, it was higher after secretion than in granules, which suggests mucins have a weakly-ordered state in granules and adopt a highly-ordered, nematic crystalline structure extracellularly. This challenges the classical view of mucus as a porous agarose-like gel and suggests an alternative model for mucin organization before and after secretion. Our study also suggests that endoplasmic reticulum stress due to molecular overcrowding contributes to mucus pathogenesis in CF cells. It encourages the development of therapeutics that target pre-secretory mechanisms in CF and other muco-obstructive lung diseases.
Defining the EM-signature of successful cell-transfection
Authors: Pemberton Joshua G. , Tenkova Tatyana , Felgner Philip , Zimmerberg Josh , Balla Tamas , Heuser John 
Published: March 2024

ABSTRACTIn this report, we describe the architecture of Lipofectamine 2000 and 3000 transfection- reagents, as they appear inside of transfected cells, using classical transmission electron microscopy (EM). We also demonstrate that they provoke consistent structural changes after they have entered cells, changes that not only provide new insights into the mechanism of action of these particular transfection-reagents, but also provide a convenient and robust method for identifying by EM which cells in any culture have been successfully transfected. This also provides clues to the mechanism(s) of their toxic effects, when they are applied in excess. We demonstrate that after being bulk-endocytosed by cells, the cationic spheroids of Lipofectamine remain intact throughout the entire time of culturing, but escape from their endosomes and penetrate directly into the cytoplasm of the cell. In so doing, they provoke a stereotypical recruitment and rearrangement of endoplasmic reticulum (ER), and they ultimately end up escaping into the cytoplasm and forming unique ’inclusion-bodies.’ Once free in the cytoplasm, they also invariably develop dense and uniform coatings of cytoplasmic ribosomes on their surfaces, and finally, they become surrounded by ’annulate’ lamellae’ of the ER. In the end, these annulate-lamellar enclosures become the ultrastructural ’signatures’ of these inclusion-bodies, and serve to positively and definitively identify all cells that have been effectively transfected. Importantly, these new EM-observations define several new and unique properties of these classical Lipofectamines, and allow them to be discriminated from other lipoidal or particulate transfection-reagents, which we find do not physically break out of endosomes or end up in inclusion bodies, and in fact, provoke absolutely none of these ’signature’ cytoplasmic reactions.