bluuebunny/crossref_metadata_2025_split huggingface dataset:
Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control
Authors: Hennigan Jennifer N. , Menacho-Melgar Romel , Sarkar Payel , Lynch Michael D. 
Published: December 2023

AbstractNanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression inE. colican be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains ofE. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, in combination with dynamic expression of chaperones and lysis enzymes needed for purification. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.
Magnetogenetic stimulation inside MRI induces spontaneous and evoked changes in neural circuits activity in rats
Authors: Chuang Kai-Hsiang , Qian Chunqi , Gilad Assaf , Pelled Galit 
Published: December 2023

AbstractThe ability to modulate specific neural circuits and simultaneously visualize and measure brain activity with MRI would greatly impact understanding brain function in health and disease. The combination of neurostimulation methods and MRI in animal models have already shown promise in elucidating fundamental mechanisms associated with brain activity. We developed an innovative magnetogenetics neurostimulation technology that can trigger neural activity through magnetic fields. Similar to other genetic-based neuromodulation methods, magnetogenetics offers cell-, area- and temporal- specific control of neural activity. However, the magnetogenetics protein (Electromagnetic Preceptive Gene (EPG)) are activated by non-invasive magnetic fields, providing a unique way to target neural circuits by the MRI gradients while simultaneously measure their effect on brain activity. EPG was expressed in rat’s visual cortex and the amplitude of low-frequency fluctuation (fALFF), resting-state functional connectivity (FC), and sensory activation was measured using a 7T MRI. The results demonstrate that EPG-expressing rats had significantly higher signal fluctuations in the visual areas and stronger FC in sensory areas consistent with known anatomical visuosensory and visuomotor connections. This new technology complements the existing neurostimulation toolbox and provides a mean to study brain function in a minimally-invasive way which was not possible previously.
The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of FA-activated proton transport
Authors: Žuna Kristina , Tyschuk Tatyana , Beikbaghban Taraneh , Sternberg Felix , Kreiter Jürgen , Pohl Elena E. 
Published: December 2023

AbstractMetabolic reprogramming in cancer cells has been linked to the mitochondrial dysfunction. Recent studies have suggested the mitochondrial 2-oxoglutarate/malate carrier (OGC) as a potential target for preventing cancer progression. Although OGC is known to be a part of the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. In this study, we investigated the contribution of recombinant murine OGC to the proton transport by measuring the conductance (Gm) of planar lipid bilayer membranes reconstituted with OGC. Our results show that OGC significantly increases Gmonly in the presence of free fatty acids (FAs) and 2,4-dinitrophenol, demonstrating for the first time its involvement in proton transport. We found that (i) the increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of FAs, and (ii) OGC substrates and inhibitors compete with FAs for the same binding site. In addition, we have identified R90 as a crucial amino acid of the binding site for FAs, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. Elucidating the contribution of OGC to the uncoupling will be crucial in the design of targeted drugs for the treatment of cancer and other metabolic diseases.
Precipitation and Extraction Methods for Protein Purification: A Meta-Analysis of Purification Performance and Cost-Effectiveness
Authors: Decker John S. , Yano Utsuki , Melgar Romel Menacho , Lynch Michael D. 
Published: December 2023

AbstractFor protein drug purification, packed-bed chromatography often remains both the predominant method and a bottleneck for cost and scalability. Accordingly, extensive efforts have been made to develop alternatives, such as precipitation and liquid-liquid extraction. Despite decades of development, such methods have been slow to see adoption in commercial processes. To diagnose the key barriers to implementation and guide future work, we have systematically reviewed studies of protein precipitation and liquid-liquid extraction. We classify the products, methods, and results of 168 publications representing 290 unique purification operations and analyze these operations in terms of both process economics and purification performance. Whereas it is generally assumed that precipitation and extraction methods will have lower costs than chromatography, we find that this is only the case under specific process conditions such as at a large manufacturing scale and low initial sample purity. Furthermore, we find that only a small number of the many precipitation and extraction methods reported to date have shown readiness for implementation in protein drug purification processes. Finally, we identify key factors governing both the economic and purification performance of this class of methods: first, that operating costs are almost entirely predictable by the ratio between the mass of phase-forming materials used and the mass of product protein yielded; second, that use of modern optimization techniques such as Design of Experiments is associated with significantly better purification performance and cost-effectiveness.HighlightsAlternative separation purification methods are not always cheaper than chromatographyThe use of a combination of phase separating agents remains largely underexplored/underutilizedLower initial purity and increasing production scale favor phase-separation over chromatographyThe direct material usage rate is an important predictor of alternative separation cost-effectivenessCurrent alternative separation method development has largely ignored optimization of direct material usage rate
Curcumin alleviates Arsenic trioxide-induced behavioural impairment, oxidative damage & morphological alterations in striatal region of mice brain
Authors: Pandey Kamlesh Kumar , Mehta Kamakshi , Kaur Balpreet , Dhar Pushpa , Kaler Saroj 
Published: December 2023

AbstractArsenic-induced neurotoxicity is well-documented in literature and reported to have dose-dependent damaging effects in the mice brain. Curcumin, a cost-effective plant polyphenol, safely demonstrates protective effects against arsenic-induced neurotoxicity by modifying oxidative stress, apoptosis, and neurochemistry in rodents’ brain.The present study determined the neuroprotective potential of curcumin (CUR) on adverse effects induced by arsenic trioxide (As2O3) in mice striatal region. Healthy adult male mice were chronically administered with varying concentrations of As2O3(2, 4 & 8 mg/kg bw) alone and along with CUR (100 mg/kg bw) via oral route for 45 days. Towards the end of experimental period, the animals were subjected to behavioural paradigm including open field task, novel object recognition, rota-rod and morris water maze. Fresh striatal tissues were collected from the animals on day 46 for biochemical analysis such as MDA, GPx and GSH. While perfusion fixed brains were processed for morphological observations.Behavioural study showed an apparent decrease in certain cognitive functions (learning and memory) and locomotor activity in mice exposed to As2O3compared to controls. Simultaneous treatment of As2O3(2, 4 & 8 mg/kg bw) and curcumin (100 mg/kg bw) alleviated theAs-induced locomotor and cognitive deficits. As2O3alone exposure also exhibited a significant increase in oxidative stress marker (MDA) and decrease in antioxidant enzyme levels (GPx, GSH). Morphological alterations were noted in mice subjected to elevated doses of As2O3(4 & 8 mg/kg bw). However, these changes were reversed in mice who received As2O3+ CUR co-treatment. Together, our findings provide preliminary evidence that curcumin protects mice striatal region from As2O3-induced behavioral, biochemical and morphological alterations.
GlnA3Mtis able to glutamylate spermine but it is not essential for the detoxification of spermine inMycobacterium tuberculosis
Authors: Krysenko Sergii , Emani Carine Sao , Bäuerle Moritz , Oswald Maria , Kulik Andreas , Meyners Christian , Hillemann Doris , Merker Matthias , Wohlers Inken , Hausch Felix , Brötz-Oesterhelt Heike , Mitulski Agnieszka , Reiling Norbert , Wohlleben Wolfgang 
Published: December 2023

ABSTRACTMycobacterium tuberculosisis well adapted to survive and persist in the infected host, escaping the host immune response. Since polyamines, which are synthesized by infected macrophages are able to inhibit the growth ofM. tuberculosis, the pathogen needs strategies to cope with toxic spermine. The actinomyceteStreptomyces coelicolor, closely related toM. tuberculosismakes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional inM. tuberculosis. In the current study we demonstrated thatM. tuberculosisgrowth was inhibited by the polyamine spermine. Using a glutamine synthetase-basedin vitroenzymatic activity assay we determined that GlnA3Mt(Rv1878) is a gamma-glutamylspermine synthetase. In anin vitrophosphate release assay we showed that purified His-Strep-GlnA3Mtas well as native GlnA3Mtprefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3Mtmay play a specific role in the detoxification of the polyamine spermine. However, the deletion of theglnA3gene inM. tuberculosisdid not result in growth inhibition or enhanced sensitivity ofM. tuberculosisin the presence of high spermine concentrations. Subsequent RNAsequencing ofM. tuberculosisbacteria revealed that the gene cluster consisting of the efflux pump-encodingrv3065-rv3066-rv3067genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations.IMPORTANCEAntibiotics for the treatment ofMycobacterium tuberculosisinfections attack classical bacterial targets, such as the cell envelope or the ribosome. UponM. tuberculosisinfection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic forM. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomyceteStreptomyces coelicolor, we hypothesized that the glutamyl-sperminesynthetase GlnA3 may be responsible for resistance against toxic spermine. Here we show that the mycobacterial glutamyl-sperminesynthetase indeed can inactivate spermine by glutamylation. However, GlnA3 is probably not the only resistance mechanism since aglnA3mutant ofM. tuberculosiscan survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3Mtas well as of the spermine transporter in the pathogenicity ofM. tuberculosisis of special interest for their validation as new targets of novel anti-tubercular drugs.
Dysregulation of innate immune signaling in animal models of Spinal Muscular Atrophy
Authors: Garcia Eric L. , Steiner Rebecca E. , Raimer Amanda C. , Herring Laura E. , Matera A. Gregory , Spring Ashlyn M. 
Published: December 2023

ABSTRACTBackgroundSpinal Muscular Atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the Survival Motor Neuron (SMN) protein. SMA presents across broad spectrum of disease severity. Unfortunately, vertebrate models of intermediate SMA have been difficult to generate and are thus unable to address key aspects of disease etiology. To address these issues, we developed aDrosophilamodel system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes.ResultsHere, we carried out transcriptomic and proteomic profiling of mild and intermediateDrosophilamodels of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the Immune Deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of an ubiquitylation complex that includes Traf6, Bendless and Diap2, and plays a pivotal role in several signaling networks.ConclusionsIn alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this processin vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.
Sporadic phage defense in epidemicVibrio choleraemediated by the toxin-antitoxin system DarTG is countered by a phage-encoded antitoxin mimic
Authors: Patel Kishen M. , Seed Kimberley D. 
Published: December 2023

AbstractBacteria and their viral predators (phages) are constantly evolving to subvert one another. Many bacterial immune systems that inhibit phages are encoded on mobile genetic elements that can be horizontally transmitted to diverse bacteria. Despite the pervasive appearance of immune systems in bacteria, it is not often known if these immune systems function against phages that the host encounters in nature. Additionally, there are limited examples demonstrating how these phages counter-adapt to such immune systems. Here, we identify clinical isolates of the global pathogenVibrio choleraeharboring a novel genetic element encoding the bacterial immune system DarTG and reveal the immune system’s impact on the co-circulating lytic phage ICP1. We show that DarTG inhibits ICP1 genome replication, thus preventing ICP1 plaquing. We further characterize the conflict between DarTG-mediated defense and ICP1 by identifying an ICP1-encoded protein that counters DarTG and allows ICP1 progeny production. Finally, we identify this protein as a functional antitoxin that abrogates the toxin DarT likely through direct interactions. Following the detection of the DarTG system in clinicalV. choleraeisolates, we observed a rise in ICP1 isolates with the functional antitoxin. These data highlight the use of surveillance ofV. choleraeand its lytic phages to understand the co-evolutionary arms race between bacteria and their phages in nature.ImportanceThe global bacterial pathogenVibrio choleraecauses an estimated 1 to 4 million cases of cholera each year. Thus, studying the factors that influence its persistence as a pathogen is of great importance. One such influence is the lytic phage ICP1, as once infected by ICP1,V. choleraeis destroyed. To date, we have observed that the phage ICP1 shapesV. choleraeevolution through the flux of anti-phage bacterial immune systems. Here, we probe clinicalV. choleraeisolates for novel anti-phage immune systems that can inhibit ICP1 and discover the toxin-antitoxin system DarTG as a potent inhibitor. Our results underscore the importance ofV. choleraeand ICP1 surveillance to elaborate novel means by whichV. choleraecan persist in both the human host and aquatic reservoir in the face of ICP1.
GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology
Authors: DiCesare Sophia M. , Ortega Antonio J. , Collier Gracen E. , Daniel Steffi , Thompson Krista N. , McCoy Melissa K. , Posner Bruce A. , Hulleman John D. 
Published: December 2023

ABSTRACTMalattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.
Advancing mRNA subcellular localization prediction with graph neural network and RNA structure
Authors: Li Fuyi , Bi Yue , Guo Xudong , Tan Xiaolan , Wang Cong , Pan Shirui 
Published: December 2023

AbstractMotivationThe asymmetrical distribution of expressed mRNAs tightly controls the precise synthesis of proteins within human cells. This non-uniform distribution, a cornerstone of developmental biology, plays a pivotal role in numerous cellular processes. To advance our comprehension of gene regulatory networks, it is essential to develop computational tools for accurately identifying the subcellular localizations of mRNAs. However, considering multi-localization phenomena remains limited in existing approaches, with none considering the influence of RNA’s secondary structure.ResultsIn this study, we propose Allocator, a multi-view parallel deep learning framework that seamlessly integrates the RNA sequence-level and structure-level information, enhancing the prediction of mRNA multi-localization. The Allocator models equip four efficient feature extractors, each designed to handle different inputs. Two are tailored for sequence-based inputs, incorporating multilayer perceptron and multi-head self-attention mechanisms. The other two are specialized in processing structure-based inputs, employing graph neural networks. Benchmarking results underscore Allocator’s superiority over state-of-the-art methods, showcasing its strength in revealing intricate localization associations.AvailabilityThe webserver of Allocator is available athttp://Allocator.unimelb-biotools.cloud.edu.au; the source code and datasets are available athttps://github.com/lifuyi774/Allocator
CCT3 drives Sorafenib resistance by inhibiting TFRC-mediated iron uptake in HCC
Authors: Zhu Huihui , Liu Qiuhong , Meng Qinna , Zhang Linjian , Lan Jiaheng , Zhu Danhua , Chen Yonxia , Aishan Nadire , Ouyang Xiaoxi , Zhang Sainan , Jin Lidan , Xiao Lanlan , Wang Linbo , Li Lanjuan , Ji Feiyang 
Published: December 2023

AbstractSorafenib is commonly utilized in the management of advanced hepatocellular carcinoma (HCC). However, its efficacy in extending patients’ survival is hindered by the development of drug resistance. By employing protein posttranslational modification (PTM) omics, including acetylome, phosphoproteome, and ubiquitinome, in conjunction with genome-wide CRISPR/Cas9 knockout library screening, we have successfully identified chaperonin containing TCP1 subunit 3 (CCT3) as a key factor contributing to Sorafenib resistance. Furthermore, we observed a reduction in the ubiquitination of CCT3 at lysine 21 (K21) subsequent to Sorafenib treatment. This study provides evidence that CCT3 hinders the recycling of transferrin receptor protein 1 (TFRC) by interacting with alpha-actinin-4 (ACTN4), which is influenced by K6-linked ubiquitination on K21. Depleting CCT3 increased the susceptibility of cells to Sorafenib-induced ferroptosis, while reintroducing CCT3 through transfection restored resistance to ferroptosis. Additionally, impairing ACTN4 or TFRC depletion compromised CCT3’s ability to inhibit Sorafenib-induced ferroptosis. In summary, targeting CCT3 presents a potential strategy for overcoming Sorafenib resistance in HCC.
Metabolic sensing in AgRP regulates sucrose preference and dopamine release in the nucleus accumbens
Authors: Reichenbach Alex , Dempsey Harry , Andrews Zane B. 
Published: December 2023

AbstractHunger increases the motivation for calorie consumption, often at the expense of low taste appeal. However, the neural mechanisms integrating calorie-sensing with increased motivation for calorie consumption remain unknown. Agouti-related peptide neurons in the arcuate nucleus of the hypothalamus sense hunger, and the ingestion of caloric solutions promote dopamine release in the absence of sweet taste perception. Therefore, we hypothesized that metabolic-sensing of hunger by AgRP neurons would be essential to promote dopamine release in the nucleus accumbens in response to caloric, but not non-caloric solutions. Moreover, we examined whether metabolic sensing in AgRP neurons affected taste preference to bitter solutions under conditions of energy need. Here we show that impaired metabolic sensing in AgRP neurons attenuated nucleus accumbens dopamine release in response to sucrose, but not saccharin, consumption. Further, metabolic sensing in AgRP neurons was essential to distinguish nucleus accumbens dopamine response to sucrose consumption when compared with saccharin. Under conditions of hunger, metabolic sensing in AgRP neurons increased the preference of sucrose solutions laced with the bitter tastant, quinine, to ensure calorie consumption whereas mice with impaired metabolic sensing in AgRP neurons maintained a strong aversion to sucrose/quinine solutions despite ongoing hunger. In conclusion, we demonstrate normal metabolic sensing in AgRP neurons drives the preference for calorie consumption, primarily when needed, by engaging dopamine release in nucleus accumbens.