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1.
Food Microbiol ; 120: 104482, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38431313

RESUMO

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.


Assuntos
Proteínas de Bactérias , Carpas , GMP Cíclico/análogos & derivados , Hafnia , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Expressão Gênica , Alimentos Marinhos , Biofilmes , Guanosina Trifosfato
2.
Mol Biol Cell ; 35(4): ar58, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38446619

RESUMO

GTPases cycle between active GTP bound and inactive GDP bound forms. Exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). GTPase activating proteins (GAPs) accelerate GTP hydrolysis, to promote the GDP bound form. We reported that the RacGEF, PIX-1, is required for assembly of integrin adhesion complexes (IAC) in striated muscle of Caenorhabditis elegans. In C. elegans, IACs are found at the muscle cell boundaries (MCBs), and bases of sarcomeric M-lines and dense bodies (Z-disks). Screening C. elegans mutants in proteins containing RhoGAP domains revealed that loss of function of rrc-1 results in loss of IAC components at MCBs, disorganization of M-lines and dense bodies, and reduced whole animal locomotion. RRC-1 localizes to MCBs, like PIX-1. The localization of RRC-1 at MCBs requires PIX-1, and the localization of PIX-1 requires RRC-1. Loss of function of CED-10 (Rac) shows lack of PIX-1 and RRC-1 at MCBs. RRC-1 exists in a complex with PIX-1. Transgenic rescue of rrc-1 was achieved with wild type RRC-1 but not RRC-1 with a missense mutation in a highly conserved residue of the RhoGAP domain. Our results are consistent with RRC-1 being a RhoGAP for the PIX pathway in muscle.


Assuntos
Caenorhabditis elegans , Proteínas Ativadoras de GTPase , Animais , Caenorhabditis elegans/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Sarcômeros/metabolismo , Guanosina Trifosfato/metabolismo , Integrinas/metabolismo
3.
Nat Commun ; 15(1): 2378, 2024 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-38493148

RESUMO

RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.


Assuntos
Proteínas , RNA Ligase (ATP) , Humanos , Guanosina Trifosfato/metabolismo , Proteínas/metabolismo , RNA/metabolismo , RNA Ligase (ATP)/genética , Splicing de RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo
4.
Protein Sci ; 33(4): e4939, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501467

RESUMO

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.


Assuntos
Neoplasias , Proteínas rho de Ligação ao GTP , Humanos , Proteínas rho de Ligação ao GTP/química , Mutação , Neoplasias/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Isoformas de Proteínas/metabolismo
5.
Nat Commun ; 15(1): 2488, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38509071

RESUMO

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATLcyto), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATLcyto can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATLcyto monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.


Assuntos
Proteínas de Drosophila , Humanos , Proteínas de Drosophila/metabolismo , Fusão de Membrana/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Hidrólise , Guanosina Trifosfato/metabolismo
6.
Science ; 383(6686): eabm9903, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38422126

RESUMO

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.


Assuntos
Bactérias , Infecções Bacterianas , Membrana Celular , Proteínas de Ligação ao GTP , Reconhecimento da Imunidade Inata , Humanos , Citocinas/química , Tomografia com Microscopia Eletrônica , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Hidrólise , Imunidade Celular , Microscopia Crioeletrônica , Gasderminas/química , Proteínas de Ligação a Fosfato/química , Conformação Proteica , Membrana Celular/química , Membrana Celular/imunologia , Caspases Iniciadoras/química , Infecções Bacterianas/imunologia , Bactérias/imunologia
7.
Cell Rep ; 43(2): 113821, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38368611

RESUMO

The titer of viruses that persist and propagate in their insect vector must be high enough for transmission yet not harm the insect, but the mechanism of this dynamic balance is unclear. Here, expression of inosine monophosphate dehydrogenase (LsIMPDH), a rate-limiting enzyme for guanosine triphosphate (GTP) synthesis, is shown to be downregulated by increased levels of N6-methyladenosine (m6A) on LsIMPDH mRNA in rice stripe virus (RSV)-infected small brown planthoppers (SBPHs; Laodelphax striatellus), the RSV vector, which decreases GTP content, thus limiting viral proliferation. Moreover, planthopper methyltransferase-like protein 3 (LsMETTL3) and m6A reader protein LsYTHDF3 are found to catalyze and recognize the m6A on LsIMPDH mRNA, respectively, and cooperate in destabilizing LsIMPDH transcripts. Co-silencing assays show that negative regulation of viral proliferation by both LsMETTL3 and LsYTHDF3 is partially dependent on LsIMPDH. This distinct mechanism limits virus replication in an insect vector, providing a potential gene target to block viral transmission.


Assuntos
Adenosina/análogos & derivados , Insetos Vetores , Animais , Guanosina Trifosfato , RNA Mensageiro/genética , Proliferação de Células
8.
J Phys Chem B ; 128(6): 1418-1427, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38323538

RESUMO

In cells, wild-type RasGTP complexes exist in two distinct states: active State 2 and inactive State 1. These complexes regulate their functions by transitioning between the two states. However, the mechanisms underlying this state transition have not been clearly elucidated. To address this, we conducted a detailed simulation study to characterize the energetics of the stable states involved in the state transitions of the HRasGTP complex, specifically from State 2 to State 1. This was achieved by employing multiscale quantum mechanics/molecular mechanics and enhanced sampling molecular dynamics methods. Based on the simulation results, we constructed the two-dimensional free energy landscapes that provide crucial information about the conformational changes of the HRasGTP complex from State 2 to State 1. Furthermore, we also explored the conformational changes from the intermediate state to the product state during guanosine triphosphate hydrolysis. This study on the conformational changes involved in the HRas state transitions serves as a valuable reference for understanding the corresponding events of both KRas and NRas as well.


Assuntos
Simulação de Dinâmica Molecular , Proteínas ras , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo
9.
Acta Crystallogr F Struct Biol Commun ; 80(Pt 3): 53-58, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38376823

RESUMO

The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.


Assuntos
GTP Fosfo-Hidrolases , Partícula de Reconhecimento de Sinal , GTP Fosfo-Hidrolases/química , Partícula de Reconhecimento de Sinal/química , Proteínas de Bactérias/química , Cristalografia por Raios X , Guanosina Trifosfato/química
10.
J Cell Biol ; 223(4)2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38323936

RESUMO

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.


Assuntos
Citoesqueleto , Guanosina Trifosfato , IMP Desidrogenase , Retina , Animais , Bovinos , Guanosina Trifosfato/biossíntese , Nucleotídeos , Fosforilação , Retina/enzimologia , IMP Desidrogenase/metabolismo
11.
J Phys Chem B ; 128(7): 1618-1626, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38351706

RESUMO

RAS is a small GTPase and acts as a binary molecular switch; the transition from its active to inactive state plays a crucial role in various cell signaling processes. Molecular dynamics simulations at the atomistic level suggest that the absence of cofactor Mg2+ ion generally leads to pronounced structural changes in the Switch-I than Switch-II regions and assists GTP binding. The presence of the Mg2+ ion also restricts the rotation of ϒ phosphate and enhances the hydrolysis rate of GTP. Further, the simulations reveal that the stability of the protein is almost uncompromised when Mg2+ is replaced with Zn2+ and not the Ca2+ ion. The specificity of H-RAS to GTP was evaluated by substituting with ATP and CTP, which indicates that the binding pocket tolerates purine bases over pyrimidine bases. However, the D119 residue specifically interacts with the guanine base and serves as one of the primary interactions that leads to the selectivity of GTP over ATP. The ring displacement of 32Y serves as gate dynamics in H-RAS which are important for its interaction with GAP for the nucleotide exchange and is restricted in the presence of ATP. Finally, the point mutations 61, 16, and 32 influence the structural changes, specifically in the Switch-II region, which are expected to impact the GTP hydrolysis and thus are termed oncogenic mutations.


Assuntos
Nucleotídeos , Proteínas , Guanosina Trifosfato/química , Ligação Proteica/genética , Proteínas/metabolismo , Hidrólise , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo
12.
Int J Mol Sci ; 25(3)2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38338709

RESUMO

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Trifosfato/metabolismo , Mutação , Proteínas ras/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Simulação de Dinâmica Molecular
13.
Molecules ; 29(3)2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38338389

RESUMO

The HRAS protein is considered a critical target for drug development in cancers. It is vital for effective drug development to understand the effects of mutations on the binding of GTP and GDP to HRAS. We conducted Gaussian accelerated molecular dynamics (GaMD) simulations and free energy landscape (FEL) calculations to investigate the impacts of two mutations (A59E and K117R) on GTP and GDP binding and the conformational states of the switch domain. Our findings demonstrate that these mutations not only modify the flexibility of the switch domains, but also affect the correlated motions of these domains. Furthermore, the mutations significantly disrupt the dynamic behavior of the switch domains, leading to a conformational change in HRAS. Additionally, these mutations significantly impact the switch domain's interactions, including their hydrogen bonding with ligands and electrostatic interactions with magnesium ions. Since the switch domains are crucial for the binding of HRAS to effectors, any alterations in their interactions or conformational states will undoubtedly disrupt the activity of HRAS. This research provides valuable information for the design of drugs targeting HRAS.


Assuntos
Simulação de Dinâmica Molecular , Transdução de Sinais , Mutação , Conformação Molecular , Guanosina Trifosfato/química , Conformação Proteica
14.
Int J Mol Sci ; 25(3)2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38338641

RESUMO

The natural cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is biosynthesized from prostaglandin E (PGE) and activated inositol phosphate (n-Ins-P), which is synthesized by a particulate rat-liver-enzyme from GTP and a precursor named inositol phosphate (pr-Ins-P), whose 5-ring phosphodiester structure is essential for n-Ins-P synthesis. Aortic myocytes, preincubated with [3H] myo-inositol, synthesize after angiotensin II stimulation (30 s) [3H] pr-Ins-P (65% yield), which is converted to [3H] n-Ins-P and [3H] cyclic PIP. Acid-treated (1 min) [3H] pr-Ins-P co-elutes with inositol (1,4)-bisphosphate in high performance ion chromatography, indicating that pr-Ins-P is inositol (1:2-cyclic,4)-bisphosphate. Incubation of [3H]-GTP with unlabeled pr-Ins-P gave [3H]-guanosine-labeled n-Ins-P. Cyclic PIP synthase binds the inositol (1:2-cyclic)-phosphate part of n-Ins-P to PGE and releases the [3H]-labeled guanosine as [3H]-GDP. Thus, n-Ins-P is most likely guanosine diphospho-4-inositol (1:2-cyclic)-phosphate. Inositol feeding helps patients with metabolic conditions related to insulin resistance, but explanations for this finding are missing. Cyclic PIP appears to be the key for explaining the curative effect of inositol supplementation: (1) inositol is a molecular constituent of cyclic PIP; (2) cyclic PIP triggers many of insulin's actions intracellularly; and (3) the synthesis of cyclic PIP is decreased in diabetes as shown in rodents.


Assuntos
Fosfatos de Inositol , Inositol , Prostaglandinas E , Humanos , Ratos , Animais , Inositol/farmacologia , Inositol/metabolismo , Fosfatos de Inositol/metabolismo , Guanosina Trifosfato , Guanosina , Fosfatos
15.
Nat Commun ; 15(1): 981, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302485

RESUMO

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.


Assuntos
Oryzias , Animais , Oryzias/genética , Segregação de Cromossomos , Centrossomo/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Vertebrados , Guanosina Trifosfato/metabolismo , Mitose
16.
Nature ; 627(8002): 212-220, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38355801

RESUMO

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.


Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular , Transporte de RNA , RNA Circular , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Carioferinas/antagonistas & inibidores , Carioferinas/deficiência , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , RNA Circular/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Proteico
17.
BMJ Health Care Inform ; 31(1)2024 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-38307617

RESUMO

OBJECTIVES: We aimed to examine the adherence of large language models (LLMs) to bacterial meningitis guidelines using a hypothetical medical case, highlighting their utility and limitations in healthcare. METHODS: A simulated clinical scenario of a patient with bacterial meningitis secondary to mastoiditis was presented in three independent sessions to seven publicly accessible LLMs (Bard, Bing, Claude-2, GTP-3.5, GTP-4, Llama, PaLM). Responses were evaluated for adherence to good clinical practice and two international meningitis guidelines. RESULTS: A central nervous system infection was identified in 90% of LLM sessions. All recommended imaging, while 81% suggested lumbar puncture. Blood cultures and specific mastoiditis work-up were proposed in only 62% and 38% sessions, respectively. Only 38% of sessions provided the correct empirical antibiotic treatment, while antiviral treatment and dexamethasone were advised in 33% and 24%, respectively. Misleading statements were generated in 52%. No significant correlation was found between LLMs' text length and performance (r=0.29, p=0.20). Among all LLMs, GTP-4 demonstrated the best performance. DISCUSSION: Latest LLMs provide valuable advice on differential diagnosis and diagnostic procedures but significantly vary in treatment-specific information for bacterial meningitis when introduced to a realistic clinical scenario. Misleading statements were common, with performance differences attributed to each LLM's unique algorithm rather than output length. CONCLUSIONS: Users must be aware of such limitations and performance variability when considering LLMs as a support tool for medical decision-making. Further research is needed to refine these models' comprehension of complex medical scenarios and their ability to provide reliable information.


Assuntos
Mastoidite , Meningites Bacterianas , Humanos , Algoritmos , Idioma , Meningites Bacterianas/tratamento farmacológico , Guanosina Trifosfato
18.
Protein Expr Purif ; 218: 106446, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38395209

RESUMO

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sefarose , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismo
19.
Cancer Lett ; 586: 216694, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38307409

RESUMO

The KRASG12D mutation was believed to be locked in a GTP-bound form, rendering it fully active. However, recent studies have indicated that the presence of mutant KRAS alone is insufficient; it requires additional activation through inflammatory stimuli to effectively drive the development of pancreatic ductal adenocarcinoma (PDAC). It remains unclear to what extent RAS activation occurs during the development of PDAC in the context of inflammation. Here, in a mouse model with the concurrent expression of KrasG12D/+ and inflammation mediator IKK2 in pancreatic acinar cells, we showed that, compared to KRASG12D alone, the cooperative interaction between KRASG12D and IKK2 rapidly elevated both the protein level and activity of KRASG12D and NRAS in a short term. This high level was sustained throughout the rest phase of PDAC development. These results suggest that inflammation not only rapidly augments the activity but also the protein abundance, leading to an enhanced total amount of GTP-bound RAS (KRASG12D and NRAS) in the early stage. Notably, while KRASG12D could be further activated by IKK2, not all KRASG12D proteins were in the GTP-bound state. Overall, our findings suggest that although KRASG12D is not fully active in the context of inflammation, concurrent increases in both the protein level and activity of KRASG12D as well as NRAS at the early stage by inflammation contribute to the rise in total GTP-bound RAS.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Mutação , Inflamação/genética , Guanosina Trifosfato
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