bluuebunny/crossref_metadata_2025_split huggingface dataset:
Silymarin Inhibits In Vitro SARS-CoV-2 Infection In Vero E6 Cells
Authors: Nnadi Nnaemeka Emmanuel , Luka Pam Dachung , Omale Simeon , Shehu Nathan Yakubu , Aguiyi John Chinyere 
Published: April 2023

AbstractThe study evaluated the invitro ability of Silymarin to inhibit SARS-COV-2 infection on Vero Cells. We set out to evaluate the hypothesis that Silymarin has both preventive and curative against SARS-COV-2. To study this, we first evaluated the safety profile of Silymarin using the Drosophila melanogaster(Harwich strain) model. Silymarin tablet film coated 140mg(Silybon-140) was used for the study. We evaluated the fly for acute toxicity, Locomotor performance, estimation of total thiol level, determination of Acetylcholinesterases (AchE) activity, Catalase activity, Glutathion-S-transferase(GST) activity and fecundity assay. To evaluate the invitro activity of Silymarin against SARS-COV-2, SARS-COV-2 isolates from oropharyngeal swabs and confirmed using qRT-PCR were cultured in Vero E6 monolayer cells. Different concentrations of silymarin concentration were used to determine pre- or post-exposure activity.The result showed that daily exposure to silymarin dose between 50% to 2000% adult dose showed no adverse effect after 28 days. Treatment of Vero cells with silymarin at the concentration 250-500ug/ml all revealed a pre-treatment effect to SARS-COV-2 in vitro and no inhibition effect was observed when the virus was first added before the addition of Silymarin. Silymarin had no adverse effect on D. melanogaster and can be used a preventive drug against SARS-COV-2
Intracellular Cytomatrix, Immobilized Biocatalysis, Matrix Micromechanics and The Warburg Effect: Entanglement of Two Age-Old Mysteries of the Normal and Malignant Cell
Authors: Shaiken Tattym E. , Siam Mohamad , Sederstrom Joel M. , Narayanan Padmini 
Published: April 2023

AbstractFor decades cancer studies have focused on molecular genetics while the role of the cytoplasm has remained obscure. Separation of the viscous fluid cytosol and elastic-solid cytomatrix has offered an opportunity to solve an age-old mystery in biochemistry; how millions of complex chemical reactions can occur simultaneously within the cell cytoplasm. The cytomatrix contains structural proteins, ribosomes, and metabolome enzymes responsible for unique biosynthetic pathways that involve immobilized biocatalysis. Immobilizing these catalytic complexes overcomes the spatial limitations for biochemical processes and allows integration of the intracellular and extracellular matrices and receptors with nuclear processes. Together, the cytosol and cytomatrix produce an interconnected synergistic network that maintains the operational flexibility of healthy cells as well as the survival of malignant cells. The cytomatrix is also responsible for cellular micromechanics and cytoplasmic motion. The combination of mechanical and biocatalytic processes triggered by extracellular signals and gene mutations in malignant cells requires additional energy. Cancer cells, consequently, utilize aerobic glycolysis, the Warburg effect, to meet the energy demands of the matrix mechanics that arise in response to imbalanced signaling and excessive biocatalytic activity. Clinical cancer is a rare event despite a high frequency of mutations, as clinical cancer is limited by the requirement for alterations that result in a high energy production state. Without these transformations, potential cancers can only survive in the quiescent state or will be eliminated. Survival of cancer cells indicates that the cancer cells were able to synchronize energy output for matrix mechanics supplying sufficient energy for tumor growth. Thus, the Warburg effect connects genetic aberrations and intracellular matrix mechanics with the ability to provide the energy supply required for the unprecedented complexity of tumor growth.
An AI-based segmentation and analysis pipeline for high-field MR monitoring of cerebral organoids
Authors: Deininger Luca , Jung-Klawitter Sabine , Richter Petra , Fischer Manuel , Karimian-Jazi Kianush , Breckwoldt Michael O. , Bendszus Martin , Heiland Sabine , Kleesiek Jens , Mikut Ralf , Hübschmann Daniel , Schwarz Daniel 
Published: April 2023

AbstractBackgroundCerebral organoids simulate the structure and function of the developing human brainin vitro, offering a large potential for personalized therapeutic strategies. The enormous growth of this research area over the past decade with its capability for clinical translation makes a non-invasive, automated analysis pipeline of organoids highly desirable.PurposeThis work presents the first application of magnetic resonance imaging (MRI) for the non-invasive quantification and quality assessment of cerebral organoids using an automated analysis tool. Three specific objectives are addressed, namely organoid segmentation to investigate organoid development over time, global cysticity classification, and local cyst segmentation.MethodsNine wildtype cerebral organoids were imaged over nine weeks using high-field 9.4T MRI including a 3D T2*-w and 2D diffusion tensor imaging (DTI) sequence. This dataset was used to train a deep learning-based 3D U-Net for organoid and local cyst segmentation. For global cysticity classification, we developed a new metric,compactness, to separate low- and high-quality organoids.ResultsThe 3D U-Net achieved a Dice score of 0.92±0.06 (mean ± SD) for organoid segmentation in the T2*-w sequence. For global cysticity classification,compactnessseparated low- and high-quality organoids with high accuracy (ROC AUC 0.98). DTI showed that low-quality organoids have a significantly higher diffusion than high-quality organoids (p < .001). For local cyst segmentation in T2*-w, the 3D U-Net achieved a Dice score of 0.63±0.15 (mean ± SD).ConclusionWe present a novel non-invasive approach to monitor and analyze cerebral organoids over time using high-field MRI and state-of-the-art tools for automated image analysis, offering a comparative pipeline for personalized medicine. We show that organoid growth can be monitored reliably over time and low- and high-quality organoids can be separated with high accuracy. Local cyst segmentation is feasible but could be further improved in the future.
High-density microbioreactor process designed for automated point-of-care manufacturing of CAR T cells
Authors: Sin Wei-Xiang , Jagannathan Narendra Suhas , Teo Denise Bei Lin , Kairi Faris , Sandikin Dedy , Cheung Ka-Wai , Lee Yie Hou , Ram Rajeev J. , Tucker-Kellogg Lisa , Birnbaum Michael E. 
Published: April 2023

AbstractWhile adoptive cell therapies have revolutionized cancer immunotherapy, current autologous chimeric antigen receptor (CAR) T cell manufacturing face challenges in scaling to meet patient demands. CAR T cell production still largely rely on fed-batch, manual, open processes that lack environmental monitoring and control, whereas most perfusion-based, automated, closed-system bioreactors currently suffer from large footprints and working volumes, thus hindering process development and scaling-out. Here, we present a means of conducting anti-CD19 CAR T cell culture-on-a-chip. We show that T cells can be activated, transduced, and expanded to densities exceeding 150 million cells/mL in a two-milliliter perfusion-capable microfluidic bioreactor, thus enabling the production of CAR T cells at clinical dose levels in a small footprint. Key functional attributes such as exhaustion phenotype and cytolytic function were comparable to T cells generated in a gas-permeable well. The process intensification and online analytics offered by the microbioreactor could facilitate high-throughput process optimization studies, as well as enable efficient scale-out of cell therapy manufacturing, while providing insights into the growth and metabolic state of the CAR T cells duringex vivoculture.
Alzheimer’s disease-specific cytokine secretion suppresses neuronal mitochondrial metabolism
Authors: Kuhn Madison K. , Fleeman Rebecca M. , Beidler Lynne M. , Snyder Amanda M. , Chan Dennis C. , Proctor Elizabeth A. 
Published: April 2023

AbstractIntroductionNeuroinflammation and metabolic dysfunction are early alterations in Alzheimer’s disease brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in Alzheimer’s disease patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear.MethodsWe used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of Alzheimer’s disease at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer.ResultsWe identified a pattern of up-regulated IFNγ, IP-10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation.ConclusionsAn Alzheimer’s disease-specific pattern of cytokine secretion reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in Alzheimer’s disease.
Rapid-kinetics degron benchmarking reveals off-target activities and mixed agonism-antagonism of MYB inhibitors
Authors: Harada Taku , Perez Monika W. , Kalfon Jérémie , Braes Flora Dievenich , Batley Rashad , Eagle Kenneth , Nabet Behnam , Leifer Becky , Kruell Jasmin , Paralkar Vikram R. , Stegmaier Kimberly , Koehler Angela N. , Orkin Stuart H. , Pimkin Maxim 
Published: April 2023

AbstractAttenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.
Real-time tracking of drug binding to Influenza A M2 reveals a high energy barrier
Authors: Tekwani Movellan Kumar , Wegstroth Melanie , Overkamp Kerstin , Leonov Andrei , Becker Stefan , Andreas Loren B. 
Published: April 2023

AbstractThe drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55-100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.Highlights5 °C kinetically traps Influenza A M2 (as dimer of dimers) in the apo formin vitro.Kinetic control allows characterization of non-specific chemical shift perturbation.Pore-bound M2 loses dissolvability in DHPC micelles, suggesting structural change.M2, residues 18-60, forms a dimer-of-dimers structure in several bilayer compositions.
Evolution of miR-155 and its probable targets:MST1R, Adam10, andCD9genes expression levels in adults and children gastritis patients withH. pyloriinfection
Authors: Mahboobi Ramina , Fallah Fatemeh , Yadegar Abbas , Shams Roshanak , Sadeghi Amir , Dara Naghi , Hakemi-Vala Mojdeh 
Published: April 2023

AbstractIntroductionH. pyloriaccounts for the main factor of gastric cancer which contributes to an immune response with the promotion of inflammation. Recently, the effect of miRNAs on the prognosis of diseases is gaining the attraction of investigators. Herein, we studied the expression levels of miR-155 and its identified targets (MST1R, Adam10, andCD9) along with miR-155 expression correlation with virulence factors ofH. pylori(vacAandcag genes),H. pyloricolonization, and inflammation, in patients’ candidates for gastric endoscopy due to gastritis.Methodsin this study total of 50 and 26 biopsy samples were taken from adults and children respectively. Biochemical and molecular identification of samples was performed using culture and PCR ofureC, 16sRNA, along with amplification ofvacAandcagAgenes for pathogenicity of bacteria. The qRT-PCR was carried out using STEM-LOOP RT-PCR (dye-based) for the evaluation of miR-155 expression level andAdam10, CD9, andMST1Rexpression levels. All Real-Time PCR reactions were carried out in triplicate and data analysis was conducted using REST.ResultsA total of 30 out of 17 biopsy samples in adults and children were positive forH. pyloriin both PCR and culture, respectively. The expression level of miR-155 is closely related to theH. pylori infectionand the down-regulation ofCD9, andMST1Rgenes inH. pylori(+) samples compared toH. pylori(-) in adults’ biopsy (p=0.0001). Although, there wasn’t any relation betweencagAandvacAgenes with the expression of miR-155 in evaluated biopsy samples in both adults and children.iConclusionThis study for the first time revealed that the expression ofMST1R, CD9, andadam10 geneswas relatively related to the expression of miR-155, and indicated that the miR-155 overexpression promoted the poor prognosis ofH. pyloriinfection in adults.
Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification
Authors: Cao Li , Do Truc , Zhu Angela D. , Alam Nathan , Link A. James 
Published: April 2023

AbstractRibosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating class of natural products of ribosomal origins. In the past decade, various sophisticated machine learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Instead, we argue that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds for novel RiPP discovery. Leveraging thatO-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized the C-terminal motif unique to RiPP-associatedO-methyltransferases as the search query to discover a novel family of RiPPs, imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, widely distributed in Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome ofNonomuraea maritima. In contrast to other RiPP associated PIMTs that recognize constrained peptides as substrates, the PIMT homolog in mNmaAMBGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. The aspartimide moiety formed is unusually stable, leading to the accumulation of the aspartimidylated productin vivo. The substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experimental validations as well as an AlphaFold model prediction. Our study suggests that PIMT-mediated aspartimide formation is an underappreciated backbone modification strategy in RiPP biosynthesis, compared to the well-studied backbone rigidification chemistries, such as thiazol(in)e and oxazol(in)e formations. Additionally, our findings suggest that aspartimide formation in Gram-positive bacterial proteomes are not limited to spontaneous protein aging and degradation.TOC Figure
Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins
Authors: Zeballos C. M. Alejandra , Moore Hayden J. , Smith Tyler J. , Powell Jackson E. , Ahsan Najah S. , Zhang Sijia , Gaj Thomas 
Published: April 2023

ABSTRACTThe TDP-43 proteinopathies, which include amyotrophic lateral sclerosis and frontotemporal dementia, are a devastating group of neurodegenerative disorders that are characterized by the mislocalization and aggregation of TDP-43. Here we demonstrate that RNA-targeting CRISPR effector proteins, a programmable class of gene silencing agents that includes the Cas13 family of enzymes and Cas7-11, can be used to mitigate TDP-43 pathology when programmed to target ataxin-2, a modifier of TDP-43-associated toxicity. In addition to inhibiting the aggregation and transit of TDP-43 to stress granules, we find that thein vivodelivery of an ataxin-2-targeting Cas13 system to a mouse model of TDP-43 proteinopathy improved functional deficits, extended survival, and reduced the severity of neuropathological hallmarks. Further, we benchmark RNA-targeting CRISPR platforms against ataxin-2 and find that high-fidelity forms of Cas13 possess improved transcriptome-wide specificity compared to Cas7-11 and a first-generation effector. Our results demonstrate the potential of CRISPR technology for TDP-43 proteinopathies.
The human acetylcholinesterase c-terminal T30 peptide activates neural growth through an alpha 7 nicotinic acetylcholine receptor mTOR pathway
Authors: Graur Alexandru , Kabbani Nadine 
Published: April 2023

AbstractAcetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contributing to development and aging. In particular, enzymatic cleavage of the carboxy terminal region of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the α7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). Proteomic analysis of cells exposed to (100nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and α7 nAChR activation. In addition, T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E, and a decrease in autophagy LC3B-II level. Taken together, our findings define mTOR as a novel pathway activated by the T30 cleavage peptide of AChE and suggest a role for mTOR signaling in cholinergic aspects of brain development, as well as disease.
Retinal input integration in excitatory and inhibitory neurons in the mouse superior colliculusin vivo
Authors: Gehr Carolin , Sibille Jérémie , Kremkow Jens 
Published: April 2023

ABSTRACTThe superior colliculus (SC) is a midbrain structure that contains one of the highest densities of inhibitory neurons in the brain and, together with the thalamocortical visual system, it plays a key role in visually guided behaviors. The SC receives direct inputs from retinal ganglion cells (RGCs) but whether excitatory and inhibitory SC neurons differentially integrate retinal activityin vivois still largely unknown. We recently established an extracellular recording approach using high-density electrodes to measure the activity of RGCs simultaneously with their postsynaptic SC targetsin vivo, that allows addressing how SC neurons integrate RGC activity. Here, we employ this method to study the functional properties and dynamics that govern retinocollicular signaling in a cell-type specific manner by identifying GABAergic SC neurons using optotagging in anesthetized VGAT- ChR2 mice. We measured 305 monosynaptically connected RGC-SC pairs, out of which approximately one third of retinal afferents connect onto inhibitory SC neurons. We show that both excitatory and inhibitory SC neurons receive comparably strong RGC inputs, with functionally similar RGC-SC pairs showing stronger connections. Our results demonstrate that similar wiring rules apply for RGCs innervation of both excitatory and inhibitory SC neurons, which is unlike the cell-type specific connectivity in the thalamocortical system. Contrasting the similar RGC-SC connection strength, we observed that RGC activity contributed more to the activity of postsynaptic excitatory SC neurons than to the activity of postsynaptic inhibitory SC neurons. This implies that the excitatory SC neurons are more specifically coupled to RGC afferent inputs, while inhibitory SC neurons may integrate additional inputs from other sources. Taken together, our study deepens the understanding of cell-type specific retinocollicular functional connectivity and emphasizes that the two major brain areas for visual processing, the visual cortex and the superior colliculus, differently integrate sensory afferent inputs.