bluuebunny/crossref_metadata_2025_split huggingface dataset:
Progressive circuit hyperexcitability in mouse neocortical slice cultures with increasing duration of activity silencing
Authors: Wise Derek L. , Greene Samuel B. , Escobedo-Lozoya Yasmin , Van Hooser Stephen D. , Nelson Sacha B. 
Published: July 2023

ABSTRACTForebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizuresin vivofollowing prolonged activity blockade. Here we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining and whole cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered three days after slices were released from five days of deprivation, they were less able to recover after ten days of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require Hebbian plasticity evoked. In single neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than seen in excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39% and 56% following five and ten days of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55% and 73% respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.SIGNIFICANCE STATEMENTWhen cortical activity is silenced during development, lifelong seizures often result. Here we explored whether these seizures result from overcompensation of homeostatic recovery mechanisms. Prior work showed that neurons briefly deprived of the ability to fire action potentials compensate by becoming more excitable, increasing synaptic drive and intrinsic excitability. We found that prolonged silencing of cortex produced a profound loss of synapse density, especially for inhibitory synapses, pointing to a circuit unable to maintain excitatory/inhibitory balance. These results show that homeostatic responses, which are normally restorative, can result in maladaptive circuit configurations when brought to extremes.
Spared nerve injury causes motor phenotypes unrelated to pain in mice
Authors: Norris Makenzie R. , Dunn Samantha S. , Aravamuthan Bhooma R. , McCall Jordan G. 
Published: July 2023

AbstractMost animal models of neuropathic pain use targeted nerve injuries quantified with motor reflexive measures in response to an applied noxious stimulus. These motor reflexive measures can only accurately represent a pain response if motor function in also intact. The commonly used spared nerve injury (SNI) model, however, damages the tibial and common peroneal nerves that should result in motor phenotypes (i.e., an immobile or “flail” foot) not typically captured in sensory assays. To test the extent of these issues, we used DeepLabCut, a deep learning-based markerless pose estimation tool to quantify spontaneous limb position in C57BL/6J mice during tail suspension following either SNI or sham surgery. Using this granular detail, we identified the expected flail foot-like impairment, but we also found SNI mice hold their injured limb closer to the body midline compared to shams. These phenotypes were not present in the Complete Freunds Adjuvant model of inflammatory pain and were not reversed by multiple analgesics with different mechanisms of action, suggesting these SNI-specific phenotypes are not directly related to pain. Together these results suggest SNI causes previously undescribed phenotypes unrelated to altered sensation that are likely underappreciated while interpreting preclinical pain research outcomes.
Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer
Authors: Baxi Aparna B. , Li Jie , Quach Vi M. , Nemes Peter 
Published: July 2023

ABSTRACTMolecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis. A comprehensive study of proteins and metabolites produced in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the rest of the embryo using liquid chromatography high-resolution mass spectrometry (HRMS). Quantification of ∼4,600 proteins and a panel of metabolites documented differential expression for ∼450 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation (OXPHOS) and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the Organizer. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO tissue. Chemical perturbation of the redox gradient affected mesoderm involution during early tissue movements of gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.
Mitochondrial-Derived Compartments Remove Surplus Proteins from the Outer Mitochondrial Membrane
Authors: Wilson Zachary N. , Balasubramaniam Sai Sangeetha , Wopat Mitchell , Hughes Adam L. 
Published: July 2023

ABSTRACTThe outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a piecemeal autophagic mechanism called themitochondrial-derivedcompartment (MDC) that removes a subset of the mitochondrial proteome. Here we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are removed by MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface.SUMMARYWilson and colleagues observe that mitochondrial-derived compartments (MDCs) selectively incorporate proteins from only the outer mitochondrial membrane (OMM), and robustly sequester both excess and mistargeted proteins into this OMM-enriched domain, suggesting MDCs act to remove surplus hydrophobic cargo from mitochondria.
Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs
Authors: Wells Michael F. , Guss Ellen J. , Zhou Hongyan , Sun Bruce , Martinez Hector ,   The NYSCF Global Stem Cell Array Team, Akopian Veronika , Noggle Scott , Paull Daniel , Moore Jennifer , Sheldon Michael , Sommer Julia E. , Benedetti Marta , Meissner Alexander , Eggan Kevin 
Published: July 2023

SUMMARYA major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.
Antimicrobial peptides and other potential biomarkers of critical illness in Sars-CoV-2 patients with acute kidney injury
Authors: dos Santos Lucas Ferreira Theotonio , Barbeiro Hermes Vieira , Barbeiro Denise Frediani , de Souza Heraldo Possolo , Pinheiro da Silva Fabiano 
Published: July 2023

AbstractAntimicrobial peptides (AMPs) are a complex network of 10-100 amino acid sequence molecules, widely distributed in Nature. Even though more than 300 AMPs have been described in mammals, cathelicidins and defensins remain the most investigated to date.Some publications examined the role of AMPs in COVID-19, but the findings are preliminary andin vivostudies are still lacking. Here, we report the plasma levels of five AMPs (LL-37, α-defensin 1, α-defensin 3, β-defensin 1 and β-defensin 3) and five cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, interleukin-10, interferon-γ and monocyte chemoattractant protein-1), in 15 healthy volunteers, 36 COVID-19 patients without Acute Kidney Injury (AKI) and 17 COVID-19 patients with AKI, since AKI is a well-known marker of worse prognosis in Sars-CoV-2 infections.We found increased levels of α-defensin 1, α-defensin 3 and β-defensin 3, but not LL-37 or β-defensin 3, in our COVID-19 population, when compared with the healthy controls, in conjunction with higher levels of interleukin-6, interleukin-10, interferon-γ and monocyte chemoattractant protein-1, putting in evidence that these AMPs and cytokines may have an important role in the systemic inflammatory response and tissue damage that characterizes severe COVID-19.Graphic Abstract
Fronto-parietal network dynamics to understand deficits in attention performance in multiple sclerosis
Authors: Welton Thomas , Meng Dewen , das Nair Roshan , Constantinescu Cris S , Auer Dorothee P , Dineen Rob A 
Published: July 2023

AbstractIntroductionImpaired attention performance is a significant burden to people with multiple sclerosis (MS). Brain connectivity fluctuates with transitions between cognitive states, so measurement of network dynamics during these conditions may help to understand MS-related attention impairment.MethodsIn people with MS and healthy controls, attention was measured using the Attention Network Test. 3T MRI was used to measure structural connectivity and both static and dynamic functional connectivity in the attention-related fronto-parietal network (FPN) at rest and during an attentionally-demanding task. Groups were compared on connectivity of the FPN during rest and task performance. Relationships between network connectivity and attention performance were tested using linear regression.ResultsThe sample comprised 37 people with MS and 23 matched controls. At rest, people with MS had significantly lower structural connectivity (R2=0.13, p=0.004), lower static functional connectivity (R2=0.07, p=0.032) and higher dynamic functional connectivity (R2=0.08, p=0.026) of the FPN. Higher dynamic connectivity was significantly associated with poorer attention performance in people with MS (R2=0.20, p=0.008). During attention-task performance, static functional connectivity was greater in people with MS than controls (R2=0.10, p=0.008). The task-induced reduction in static connectivity (relative to rest) was directly related to attention performance (R2=0.23, p<0.001).ConclusionIncreased dynamic functional connectivity of the FPN at rest may be a useful indicator of deficits in sustained attention in people with MS. The transition from rest to active-attentive state is accompanied by an increase in dynamic connectivity, and decrease in static connectivity which may be helpful in understanding aetiology and treatment of attention impairment.
Performance of BD Onclarity HPV assay on FLOQSwabs vaginal self-samples
Authors: Martinelli Marianna , Latsuzbaia Ardashel , Bonde Jesper , Pedersen Helle , Iacobone Anna D. , Bottari Fabio , Piana Andrea F. , Pietri Roberto , Cocuzza Clementina E. , Arbyn Marc ,   Extended Valhudes Study Group
Published: July 2023

AbstractThis study assessed the accuracy of hrHPV testing of BD Onclarity™ HPV (Onclarity) assay on vaginal self-collected FLOQSwab® versus cervical samples to ensure similar accuracy to detect cervical intraepithelial neoplasia. Testing was performed on two automated platforms, BD Viper™ and BD COR™, to evaluate the effect of machine and using two vaginal self-samples to analyse the influence of collection, transport, and freezing-unfreezing on the results.A cervical sample and two self-samples were collected from 300 women. The first collected vaginal and the cervical sample were tested on BD Viper™ and the second swab was frozen and subsequently tested on both automated systems. Test results on vaginal and cervical specimens were considered the index and comparator, respectively; colposcopy and histology were reference standards.Relative sensitivity for ≥CIN2 on vaginal samples analysed using the three different workflows vs the cervical sample was 1.01 (0.97-1.06), 1.01 (0.97-1.06), and 1.00 (0.95-1.05), respectively.Relative specificity resulted 0.83 (0.73-0.94), 0.76 (0.67-0.87) and 0.82 (0.73-0.92) for the 1st, 2nd self-collected sample tested on BD VIPER™ and 2nd self-collected sample tested on BD COR™.Cut-off optimization for HPV positivity defined at Ct ≤38.3 for HPV16, ≤34.2 for HPV18 and ≤31.5 for all other types showed an increased relative specificity with similar sensitivity. No significant difference was observed between self-samples tested with the two platforms and between first and second-collected swabs.Onclarity assay on FLOQSwab®using both platforms showed similar sensitivity but lower specificity to detect ≥CIN2 compared to cervical samples. By cut-off optimization, non-inferior specificity could be reached.
A systems-level analysis of the mutually antagonistic roles of RKIP and BACH1 in dynamics of cancer cell plasticity
Authors: Shyam Sai , Soundharya R , Sehgal Manas , Jolly Mohit Kumar 
Published: July 2023

AbstractPhenotypic plasticity is a hallmark of cancer metastasis. Epithelial-mesenchymal transition (EMT) is an important axis of phenotypic plasticity. Raf kinase-B inhibitor protein (RKIP) and BTB and CNC homology 1 (BACH1) are two proteins reported to influence EMT. In breast cancer, they act antagonistically, but the exact nature of their roles in mediating EMT and associated other axes of plasticity remains unclear. Here, analysing transcriptomic data, we reveal their antagonistic trends in a pan-cancer manner, in terms of association with EMT, metabolic reprogramming and immune evasion via PD-L1. Next, we developed and simulated a mechanism-based gene regulatory network that captures how RKIP and BACH1 engage in feedback loops with drivers of EMT and stemness. We found that RKIP and BACH1 belong to two separate “teams” of players – while BACH1 belonged to the one that drove pro-EMT, stem-like and therapy-resistant cell-states, RKIP is a member of a team that enables pro-epithelial, less stem-like and therapy-sensitive phenotypes. Finally, we observed that low RKIP levels and concomitant upregulated BACH1 levels associated with worse clinical outcomes in many cancer types. Together, our systems-level analysis indicates that the emergent dynamics of underlying regulatory network underlie the antagonistic patterns of RKIP and BACH1 with various axes of cancer cell plasticity, as well as with patient survival data.
The Use of Fluid-phase 3D Printing to Pattern Alginate-gelatin Hydrogel Properties to Guide Cell Growth and BehaviourIn Vitro
Authors: Andrea Souza , Kevin McCarthy , Brian J. Rodriguez , Emmanuel G Reynaud 
Published: July 2023

ABSTRACT3D (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is in its infancy and (bio)printing biomimetic constructions for tissue formationin vitrois still a default. As a new methodology to improvein vitrostudies, we suggest the use of a cross-linkable aqueous support bath to pattern the characteristics of the scaffolds during the 3D printing process. Using fluid-phase, different molecules can be added to specific locations of the substrate promoting cell behaviour guidance and compartmentalization. Moreover, mechanical aspects can be customized by changing the type or concentration of the solution in which the (bio)printing is acquired. In this study, we first assessed different formulations of alginate/gelatin to improve cell colonization in our printings. On formulations with lower gelatin content, the U2OS cells increased 2.83 times the cell growth. In addition, the alginate-gelatin hydrogel presented a good printability in both air and fluid-phase, however the fluid-phase printings showed better printing fidelity as it diminished the collapsing and the spreading of the hydrogel strand. Next, the fluid-phase methodology was used to guide cell colonization in our printings. First, different stiffness were created by crosslinking the hydrogel with different concentrations of CaCl2during the printing process. As a result, the U2OS cells were compartmentalized on the stiffer parts of the printings. In addition, using fluid-phase to add RGD molecules to specific parts of the hydrogel has also promoted guidance on cell growth. Finally, our results showed that by combining stiffer alginate-gelatin hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell growth by up to 3.17-fold. This work presents a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate a more faithful replication of thein vivoenvironment.
Drosophila Amus and Bin3 methylases functionally replace mammalian MePCE for capping and the stabilization of U6 and 7SK snRNAs
Authors: Peng Qiu , Wang Yiqing , Xiao Ying , Chang Hua , Luo Shishi , Wang Danling , Rong Yikang S. 
Published: July 2023

AbstractU6 and 7SK snRNAs process a 5’ cap, believed to be essential for their stability and maintained by mammalian MePCE or Drosophila Bin3 enzymes. Although loss of either protein results in 7SK instability, loss of neither is associated with U6 reduction. Their yeast homolog is also not required for U6 stability, casting further doubts on the function of capping U6. Here we show that the Drosophila Amus protein, homologous to both Bin3 and MePCE, is essential for U6 but not 7SK stability. A full function of Amus is required for Drosophila development, and that rests primarily on Amus’s methylase activity. Remarkably, the loss of U6 is rescued by the expression of an Amus-MePCE hybrid protein harboring the methyltransferase domain from MePCE, highlighting the conserved function of the two proteins as the U6 capping enzyme. Our new investigations in human cells establish a dependence of both U6 and 7SK stability on MePCE, resolving a long-standing uncertainty. While uncovering an interesting division of labor of Bin3/MePCE/Amus proteins, we discovered a “Bin3-Box” domain present only in enzymes associated with 7SK regulation. Targeted mutagenesis in Drosophila confirmed its importance for Bin3 function, revealing a possible conserved element in 7SK but not U6 biology.
Multivalent dynamic colocalization of avian influenza polymerase and nucleoprotein by intrinsically disordered ANP32A reveals the molecular basis of human adaptation
Authors: Camacho-Zarco Aldo R. , Yu Lefan , Krischuns Tim , Dedeoglu Selin , Maurin Damien , Bouvignies Guillaume , Crépin Thibaut , Ruigrok Rob W.H. , Cusack Stephan , Naffakh Nadia , Blackledge Martin 
Published: July 2023

ABSTRACTAdaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remain poorly understood. Here we characterise ternary complexes formed between ANP32A, FluPol and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted Pol, containing the E627K mutation, simultaneously bind the identical extended linear motif on human ANP32A, in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.