bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered calcium responses and antioxidant properties in Friedreich’s ataxia-like cerebellar astrocytes
Authors: Marullo Chiara , Croci Laura , Giupponi Iris , Rivoletti Claudia , Zuffetti Sofia , Bettegazzi Barbara , Casoni Filippo , Cremona Ottavio , Consalez Gian Giacomo , Codazzi Franca 
Published: July 2024

ABSTRACTFriedreich’s ataxia (FRDA) is a neurodegenerative disorder characterized by severe neurological signs affecting both the peripheral and central nervous system, caused by reduced levels of the frataxin protein (FXN). While several studies highlight cellular dysfunctions in neurons and various other cell types, there is limited information on the effects of FXN depletion in astrocytes and on the potential non-cell autonomous mechanisms affecting neurons in FRDA. In this study, we generated a model of FRDA cerebellar astrocytes to unveil phenotypic alterations that might contribute to cerebellar atrophy and the degeneration of glutamatergic neurons observed in cerebellar dentate nuclei. We treated primary cerebellar astrocytes with an RNA interference-based approach, to achieve a reduction of FXN comparable to that observed in patients. These FRDA-like astrocytes display some typical features of the disease, such as an increase of oxidative stress, as well as specific functional alterations. Notably, cerebellar astrocytes deplete their reduced glutathione content, becoming more susceptible to oxidative insults. Moreover, FRDA-like astrocytes exhibit alterations of calcium homeostasis, with a reduction in calcium content in the intracellular stores and a corresponding change of calcium responses to purinergic stimuli. Our findings shed light on cellular changes caused by FXN downregulation in cerebellar astrocytes, which can interfere with their physiological and complex interaction with neurons. The potentially impaired ability to provide neuronal cells with glutathione or to release neuromodulators and bioactive molecules in a calcium-dependent manner could impact neuronal function and contribute to neurodegeneration.
Revisiting Sodium Phosphotungstate and Ammonium Molybdate as non-radioactive negative staining agents for single particle analysis
Authors: Gunkel Monika , Macha Arthur , Behrmann Elmar 
Published: July 2024

AbstractThis study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate for negative staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step both stains yield comparable images to uranyl formate. Subsequently, using β-galactosidase, it is shown that both stains can also successfully be employed for single particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are non-radioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample grid preparation – thus directly after purification – accessible to a broader range of scientists.
Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing
Authors: Chey Yu C. J. , Gierus Luke , Lushington Caleb , Arudkumar Jayshen C. , Geiger Ashleigh , Staker Lachlan G. , Robertson Louise J. , Pfitzner Chandran , Kennedy Jesse G. , Lee Ryan H. B. , Godahewa Gelshan I. , Thomas Paul Q. , Adikusuma Fatwa 
Published: July 2024

AbstractEnsuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.
Developmental timecourse of aptitude for motor skill learning in mouse
Authors: Kim Taehyeon , Hooks Bryan M. 
Published: July 2024

AbstractLearning motor skills requires plasticity in the primary motor cortex (M1). But the capacity for cortical circuit plasticity varies over developmental age in sensory cortex. This study assesses the normal developmental trajectory of motor learning to assess how aptitude might vary with age. We trained mice of both sexes to run on a custom accelerating rotarod at ages from postnatal day (P) 20 to P120, tracking paw position and quantifying time to fall and changes in gait pattern. While animals of all ages were able to perform better after five training sessions, performance improved most rapidly on the first training day for mice between ages P30-60, suggesting an age with heightened plasticity. Learning this task required M1, because pharmacological inactivation of M1 prevented improvement in task performance. Paw position and gait patterns changed with learning, though differently between age groups. Successful mice learned to shift their gait from hopping to walking. Notably, this shift in gait happened earlier in the trial for forelimbs in comparison to hindlimbs. Thus, motor plasticity might more readily occur in forelimbs. Changes in gait and other kinematic parameters are an additional learning metric beyond time to fall, offering insight into how mice improve performance. Overall, these results suggest mouse motor learning has a developmental trajectory.SignificancePlasticity in sensory cortex is restricted to a limited developmental window. Learning motor skills requires motor cortex plasticity, but it is unknown whether learning aptitude changes over development. Here, we define the developmental trajectory of motor learning aptitude for the accelerating rotarod task in mice, demonstrating a sensitive period for motor learning. Learning peaks at P30-P60, with mice learning to shift from hopping to walking gait to stay on the rotarod longer. Learning this task depends on M1. Further, the gait shift in forelimbs precedes hindlimbs. Knowing the peak in motor plasticity identifies the time window at which we should seek to understand the circuit basis of motor learning plasticity in cortex.
Long range regulation of transcription scales with genomic distance in a gene specific manner
Authors: Jensen Christina L , Chen Liang-Fu , Swigut Tomek , Crocker Olivia J , Yao David , Bassik Mike C , Ferrell James E , Boettiger Alistair N , Wysocka Joanna 
Published: July 2024

SummaryWhile critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across thePrdm8-Fgf5locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.
CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION
Authors: Akshita Chhabra , Christian Hoffmann , Aleksandr Korobeinikov A. , Jakob Rentsch , Linda Kokwaro , Luka Gnidovec , Cristina Román-Vendrell , Emma Johnson C. , Jaqulin Wallace N. , Branislava Rankovic , Eleonora Perego , Sarah Köster , Silvio Rizzoli O. , Helge Ewers , Jennifer Morgan R. , Dragomir Milovanovic 
Published: July 2024

AbstractNeuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation (LLPS). This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. The reorganization of SVs, synapsins and actin is a hallmark of synaptic activity, but their interplay is still unclear. Here we combined the reconstitution approaches and super-resolution imaging to dissect the roles of synapsin-SV condensates in the organization of the presynaptic actin cytoskeleton. Our data indicate that LLPS of synapsin initiates actin polymerization, allowing for SV:synapsin:actin assemblies to facilitate the mesoscale organization of SV clusters along axons mimicking the native presynaptic organization in both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-SVs condensates is an essential building block on a roadmap to unravel how coordinated neurotransmission along the axon enables circuit function and behavior.
Lipotoxicity Induces Beta Cell Small Extracellular Vesicle-mediated β-cell Dysfunction
Authors: Roy Abhishek , Hoff Alexandra , Her Tracy K. , Ariyaratne Gallage , Rajagopalan Kamalnath Sankaran , Brown Matthew R. , Soto-González Alondra , Matveyenko Aleksey V. , Javeed Naureen 
Published: July 2024

AbstractChronically elevated circulating excess free fatty acids (i.e.lipotoxicity) is a pathological process implicated in several metabolic disorders, including obesity-driven Type 2 diabetes (T2D). Lipotoxicity exerts detrimental effects on pancreatic islet β-cells by reducing glucose-stimulated insulin secretion (GSIS), altering β-cell transcriptional identity, and promoting apoptosis. While β-cell-derived small extracellular vesicles (sEV) have been shown to contribute to β-cell failure in T2D, their specific role in lipotoxicity-mediated β-cell failure remains to be elucidated. In this work, we demonstrate that lipotoxicity enhances the release of sEVs from β-cells, which exhibit altered proteomic and lipidomic profiles. These lipotoxic sEV induce β-cell dysfunction in healthy mouse and human islets and trigger significant islet transcriptional changes, including the upregulation of genes associated with the TGFβ/Smad3 pathway, as noted by RNA sequencing. Importantly, pharmacological inhibition of the TGFβI/II receptor improved lipotoxic sEV-induced β-cell dysfunction, underscoring their involvement in activating the TGFβ/Smad3 pathway during this process. We have comprehensively characterized lipotoxic β-cell sEVs and implicated their role in inducing β-cell functional failure in T2D. These findings highlight potential avenues for therapeutic interventions targeting sEV-mediated pathways to preserve β-cell health in metabolic disorders.Article HighlightsDiabetogenic lipotoxic conditions enhance β-cell sEV release and induce alterations in both protein and lipid content.Global islet transcriptional changes and alterations in β-cell function were noted upon exposure to lipotoxic sEV.Lipotoxic sEV were shown to activate the TGFβ/Smad3 pathway and blockade of this pathway improved β-cell function.
Biguanides Associate with Decreased Early Mortality and Risk of Acute Kidney Injury In Hospitalized COVID-19 Patients: a nationwide retrospective cohort study in Japan
Authors: Sugimoto Mari , Kikuchi Hiroaki , Sohara Eisei , Limbutara Kavee , Hirakawa Akihiro , Mori Takayasu , Susa Koichiro , Oya Shuichiro , Suzuki Takefumi , Iimori Soichiro , Fushimi Kiyohide , Uchida Shinichi 
Published: July 2024

AbstractBackgroundBiguanide (BG) is the most-prescribed oral glucose-lowering medication worldwide and has potential for further therapeutic applications. The Coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency. Nevertheless, there are still no established low-cost treatments against COVID-19, of which the morbidity and mortality rates varing from country to country. Therefore, a nationwide study of the COVID-19 affected population is essential to explore therapeutic effect of BG against COVID-19.MethodsFrom the inpatient databases in Japan, covering the period from September 2021 to March 2023, which encompasses the era following the development of COVID-19 vaccines, we extracted data of 168,370 COVID-19 patients aged 20 to under 80 years who were suffered from diabetes mellitus treated with oral antidiabetic agents. The primary outcome was 100-day in-hospital mortality, and secondary outcome was the incidence of acute kidney injury (AKI) during hospitalization. We compared outcomes in patients who received BG with those in patients who did not, using a logistic regression analysis and Cox proportional hazards under both propensity score-unmatched and matched cohorts.ResultsThe incidence of in-hospital death was significantly lower in the BG group (1.18 %) compared to the non-BG group (2.41%) (p < 0.001). Similarly, the incidence of AKI during hospitalization was significantly lower in the BG group (0.66 %) compared to the non-BG group (1.12%) (p < 0.001). Kaplan-Meier analysis from the propensity-score matched cohort showed a significantly better survival rate in the BG group (adjusted HR, 0.580; 95% CI 0.510-0.658; p < 0.001).ConclusionIn COVID-19 patients, the use of oral biguanide use may be associated with a reduced in-hospital mortality and risk of AKI.
Pyramidal neurons proportionately alter the identity and survival of specific cortical interneuron subtypes
Authors: Wu Sherry Jingjing , Dai Min , Yang Shang-Po , McCann Cai , Qiu Yanjie , Marrero Giovanni J. , Stogsdill Jeffrey A. , Di Bella Daniela J. , Xu Qing , Farhi Samouil L. , Macosko Evan Z. , Chen Fei , Fishell Gord 
Published: July 2024

AbstractThe mammalian cerebral cortex comprises a complex neuronal network that maintains a delicate balance between excitatory neurons and inhibitory interneurons. Previous studies, including our own research, have shown that specific interneuron subtypes are closely associated with particular pyramidal neuron types, forming stereotyped local inhibitory microcircuits. However, the developmental processes that establish these precise networks are not well understood. Here we show that pyramidal neuron types are instrumental in driving the terminal differentiation and maintaining the survival of specific associated interneuron subtypes. In a wild-type cortex, the relative abundance of different interneuron subtypes aligns precisely with the pyramidal neuron types to which they synaptically target. InFezf2mutant cortex, characterized by the absence of layer 5 pyramidal tract neurons and an expansion of layer 6 intratelencephalic neurons, we observed a corresponding decrease in associated layer 5b interneurons and an increase in layer 6 subtypes. Interestingly, these shifts in composition are achieved through mechanisms specific to different interneuron types. While SST interneurons adjust their abundance to the change in pyramidal neuron prevalence through the regulation of programmed cell death, parvalbumin interneurons alter their identity. These findings illustrate two key strategies by which the dynamic interplay between pyramidal neurons and interneurons allows local microcircuits to be sculpted precisely. These insights underscore the precise roles of extrinsic signals from pyramidal cells in the establishment of interneuron diversity and their subsequent integration into local cortical microcircuits.
Mathematical modeling using box-behnken response surface design for optimal DNA yield with variable parameters of time, temperature, and proteinase K concentration
Authors: Afzal Farwa , Zia Saeeda , Saif Rashid 
Published: July 2024

AbstractThe current research aims to explore an enzyme-assisted inorganic DNA extraction protocol to obtain maximum yield from whole blood. We considered various operating conditions such as incubation temperature and time along with proteinase K enzyme concentration. To model and simulate the extraction process, Box-Behnken Design (BBD) was employed with three factors and its five levels, coupled with the desired functional response surface methodology (RSM). RSM entails fitting a mathematical model for experimental data to optimize a response variable with Box-Behnken Design. The best simulation settings yielded the maximum DNA at an incubation temperature of 56°C for 10 hours and proteinase K enzyme concentration of 10µL. Utilizing these parameters, a yield of 300µL was obtained. Furthermore, other DNA extraction parameters may also be added for a more robust design for obtaining the maximum DNA yield.
A multivalent RSV vaccine based on the Modified Vaccinia Ankara vector shows moderate protection against disease caused by RSV in older adults in a Phase 3 clinical study
Authors: Jordan Elke , Jenkins Victoria , Silbernagl Günter , Velasco Chávez Maria Paulina , Schmidt Darja , Schnorfeil Frauke , Schultz Stephanie , Chen Liddy , Salgado Fernanda , Jacquet Jeanne-Marie , Welte Tobias , De Moerlooze Laurence 
Published: July 2024

ABSTRACTRespiratory syncytial virus (RSV) causes a significant disease burden in older adults. The live recombinant vaccine based on a nonreplicating modified vaccinia Ankara (MVA-BN) poxvirus, MVA-BN-RSV, encoding for multiple proteins of RSV subtypes A and B, was assessed for efficacy against respiratory disease caused by RSV.Adults aged ≥ 60 years, with or without underlying chronic conditions, were enrolled and randomized in a 1:1 ratio to receive a single dose of vaccine or placebo and were followed for disease caused by RSV infection during the 2022-2023 season. The 2 primary endpoints were RSV-associated lower respiratory tract disease (LRTD) with ≥ 3 and ≥ 2 symptoms; acute respiratory disease (ARD) was a key secondary endpoint. The humoral RSV-specific immune response was assessed at baseline and 14 days post-vaccination. Safety was evaluated by collection of solicited adverse events (AEs) and unsolicited AEs for 7 and 28 days post- vaccination respectively, and SAEs for the entire study period.In total, 18348 participants were included in the final efficacy and safety analyses. Vaccine efficacy was 42.9% (95% CI: -16.1; 71.9) against RSV-associated LRTD with ≥ 3 symptoms, 59.0% (95% CI: 34.7; 74.3) against LRTD with ≥ 2 symptoms, and 48.8% (95% CI: 25.8; 64.7) against ARD. The primary objective was not met for LRTD with ≥ 3 symptoms since the lower bound of the 95% CI was below 20%, the prespecified success criterion. The vaccine-elicited immune response showed mean fold-increases of 1.7 for RSV A and B neutralizing antibodies and 2.9 and 4.3 for RSV-specific IgG and IgA, respectively. The vaccine displayed mild to moderate reactogenicity, and no safety concerns were identified.MVA-BN-RSV induced suboptimal protection against RSV-associated LRTD, likely due to suboptimal neutralizing antibody response. The vaccine had an acceptable safety profile and confirmed immunogenicity, overall showing promise for MVA-BN-vectored constructs targeting other diseases.
Determinants of Timely Malaria Treatment among Under-Five Children Attending Public Health Facilities in Kisumu East Sub-County, Kenya: A Health Facility-Based Cross-Sectional Study
Authors: Ochieng Geofrey , Sampa Mutale , Maritim Patricia , Silumbwe Adam , Zulu Joseph M , Kato Joseph , Jacobs Choolwe 
Published: July 2024

ABSTRACTEvidence shows that most child malaria deaths occur at home shortly after symptom onset, highlighting the need for timely treatment. This study aimed to assess the determinants of timely malaria treatment among under-five children who receive care at public health facilities in Kisumu East sub-county, Kenya.A health facility-based cross-sectional study was conducted in Kisumu East sub-county, Kenya, between 5thApril and 26thMay 2023. The study employed a two-stage stratified-cluster sampling method, first selecting hospitals and then using systematic sampling to select caregivers. Data collection was done electronically using structured questionnaires. Associations at bivariable level were assessed using either the Chi-square or Fisher’s exact test based on assumptions. Multiple logistic regression with robust standard errors was applied at a 5% significance level to establish determinants of timely malaria treatment among under-five children. STATA version 16 (College Station, Texas 77845 USA) was used for all analyses.The sample included a total of 434 caregivers of under-five children. The study revealed that caregivers’ ability to recognize malaria symptoms was associated with higher odds of seeking timely malaria treatment for their children (AOR=2.92; 95% CI=1.36-6.25; p=0.006). Additionally, having health insurance cover was associated with higher odds of timely treatment (AOR=2.12; 95% CI=1.25-3.59; p=0.005). Those who visited herbalists before seeking care were less likely to seek timely treatment (AOR=0.13; 95% CI=0.05-0.34; p<0.0001). Fear of drugs’ side effects reduced the odds of timely treatment (AOR=0.50; 95% CI=0.29-0.87; p=0.013).Timely malaria treatment was linked to the ability to tell symptoms and having health insurance, while delayed malaria treatment was related to herbalist visits and fear of malaria, among others. There is need for relevant stakeholders to implement strategies that address misconceptions about drug side effects, offer affordable insurance, integrate the informal health sector, and educate caregivers about under-five malaria symptoms in order to promote timely malaria treatment.