RESUMO
The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
Assuntos
Processamento Alternativo , Isoformas de RNA , Sítio de Iniciação de Transcrição , Humanos , Poliadenilação , Regiões Promotoras Genéticas , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
Diet modulates the gut microbiome, which in turn can impact the immune system. Here, we determined how two microbiota-targeted dietary interventions, plant-based fiber and fermented foods, influence the human microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study (n = 18/arm) combined with -omics measurements of microbiome and host, including extensive immune profiling, we found diet-specific effects. The high-fiber diet increased microbiome-encoded glycan-degrading carbohydrate active enzymes (CAZymes) despite stable microbial community diversity. Although cytokine response score (primary outcome) was unchanged, three distinct immunological trajectories in high-fiber consumers corresponded to baseline microbiota diversity. Alternatively, the high-fermented-food diet steadily increased microbiota diversity and decreased inflammatory markers. The data highlight how coupling dietary interventions to deep and longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Fermented foods may be valuable in countering the decreased microbiome diversity and increased inflammation pervasive in industrialized society.
Assuntos
Dieta , Microbioma Gastrointestinal , Imunidade , Biodiversidade , Fibras na Dieta/farmacologia , Comportamento Alimentar , Feminino , Alimentos Fermentados , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacosRESUMO
Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.
Assuntos
Diarreia/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Animais , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Ceco/química , Ceco/metabolismo , Ceco/microbiologia , Ceco/patologia , Colo/química , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Diarreia/imunologia , Diarreia/microbiologia , Diarreia/veterinária , Fezes/microbiologia , Glicosídeo Hidrolases/metabolismo , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Metagenômica , Camundongos , Concentração Osmolar , Polietilenoglicóis/metabolismo , Proteoma/análise , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Verrucomicrobia/efeitos dos fármacos , Verrucomicrobia/genética , Verrucomicrobia/isolamento & purificaçãoRESUMO
Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. Here, we identify a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM(+) neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny.
Assuntos
Drosophila melanogaster/fisiologia , Neurônios/metabolismo , Taquicininas/metabolismo , Agressão , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Masculino , Mutação , Receptores de Taquicininas/genética , Receptores de Taquicininas/metabolismo , Caracteres SexuaisRESUMO
Polarized (sub)millimetre emission from dust grains in circumstellar disks was initially thought to be because of grains aligned with the magnetic field1,2. However, higher-resolution multi-wavelength observations3-5 and improved models6-10 found that this polarization is dominated by self-scattering at shorter wavelengths (for example, 870 µm) and by grains aligned with something other than magnetic fields at longer wavelengths (for example, 3 mm). Nevertheless, the polarization signal is expected to depend on the underlying substructure11-13, and observations until now have been unable to resolve polarization in multiple rings and gaps. HL Tau, a protoplanetary disk located 147.3 ± 0.5 pc away14, is the brightest class I or class II disk at millimetre-submillimetre wavelengths. Here we show deep, high-resolution polarization observations of HL Tau at 870 µm, resolving polarization in both the rings and the gaps. We find that the gaps have polarization angles with a notable azimuthal component and a higher polarization fraction than the rings. Our models show that the disk polarization is due to both scattering and emission from the aligned effectively prolate grains. The intrinsic polarization of aligned dust grains is probably more than 10%, which is much higher than that expected in low-resolution observations (about 1%). Asymmetries and dust features that are not seen in non-polarimetric observations are seen in the polarization observations.
RESUMO
The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
The social system of animals involves a complex interplay between physiology, natural history, and the environment. Long relied upon discrete categorizations of "social" and "solitary" inhibit our capacity to understand species and their interactions with the world around them. Here, we use a globally distributed camera trapping dataset to test the drivers of aggregating into groups in a species complex (martens and relatives, family Mustelidae, Order Carnivora) assumed to be obligately solitary. We use a simple quantification, the probability of being detected in a group, that was applied across our globally derived camera trap dataset. Using a series of binomial generalized mixed-effects models applied to a dataset of 16,483 independent detections across 17 countries on four continents we test explicit hypotheses about potential drivers of group formation. We observe a wide range of probabilities of being detected in groups within the solitary model system, with the probability of aggregating in groups varying by more than an order of magnitude. We demonstrate that a species' context-dependent proclivity toward aggregating in groups is underpinned by a range of resource-related factors, primarily the distribution of resources, with increasing patchiness of resources facilitating group formation, as well as interactions between environmental conditions (resource constancy/winter severity) and physiology (energy storage capabilities). The wide variation in propensities to aggregate with conspecifics observed here highlights how continued failure to recognize complexities in the social behaviors of apparently solitary species limits our understanding not only of the individual species but also the causes and consequences of group formation.
Assuntos
Carnívoros , Comportamento Social , Animais , Carnívoros/fisiologiaRESUMO
We present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.
Assuntos
DNA , Glicerol , Conformação de Ácido Nucleico , Pirenos , DNA/química , Guanina , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pirenos/química , Glicerol/análogos & derivados , Glicerol/químicaRESUMO
Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F H2N and intramolecular 3'H N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.
Assuntos
DNA Forma Z , Conformação de Ácido Nucleico , DNA Forma Z/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Halogenação , Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Concentração Osmolar , Ressonância Magnética Nuclear Biomolecular , DNA de Forma B/química , Modelos Moleculares , DNA/química , DNA/metabolismoRESUMO
The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.
Assuntos
Citosina , DNA , Pareamento de Bases , Citosina/análogos & derivados , DNA/química , Conformação de Ácido Nucleico , Oxazinas/química , Oxazinas/metabolismo , Células HeLa , Humanos , FluorescênciaRESUMO
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22 , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Humanos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Mutação com Ganho de Função , Linfócitos T/metabolismo , Linfócitos T/imunologia , Células Jurkat , Células HEK293RESUMO
Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.
Assuntos
Oligonucleotídeos Antissenso , Oligonucleotídeos Fosforotioatos , Humanos , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Antissenso/química , DNA , Transporte Biológico , EnxofreRESUMO
Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.
Assuntos
Canais Iônicos , Células Supressoras Mieloides , Prótons , Linfócitos T , Animais , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Células Supressoras Mieloides/imunologia , NADPH Oxidase 2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologiaRESUMO
BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.
Assuntos
Camundongos Knockout , Vesículas Sinápticas , Animais , Camundongos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
OBJECTIVE: Patients with alcohol-associated hepatitis (AH) have a high mortality. Alcohol exacerbates liver damage by inducing gut dysbiosis, bacterial translocation and inflammation, which is characterised by increased numbers of circulating and hepatic neutrophils. DESIGN: In this study, we performed tandem mass tag (TMT) proteomics to analyse proteins in the faeces of controls (n=19), patients with alcohol-use disorder (AUD; n=20) and AH (n=80) from a multicentre cohort (InTeam). To identify protein groups that are disproportionately represented, we conducted over-representation analysis using Reactome pathway analysis and Gene Ontology to determine the proteins with the most significant impact. A faecal biomarker and its prognostic effect were validated by ELISA in faecal samples from patients with AH (n=70), who were recruited in a second and independent multicentre cohort (AlcHepNet). RESULT: Faecal proteomic profiles were overall significantly different between controls, patients with AUD and AH (principal component analysis p=0.001, dissimilarity index calculated by the method of Bray-Curtis). Proteins that showed notable differences across all three groups and displayed a progressive increase in accordance with the severity of alcohol-associated liver disease were predominantly those located in neutrophil granules. Over-representation and Reactome analyses confirmed that differentially regulated proteins are part of granules in neutrophils and the neutrophil degranulation pathway. Myeloperoxidase (MPO), the marker protein of neutrophil granules, correlates with disease severity and predicts 60-day mortality. Using an independent validation cohort, we confirmed that faecal MPO levels can predict short-term survival at 60 days. CONCLUSIONS: We found an increased abundance of faecal proteins linked to neutrophil degranulation in patients with AH, which is predictive of short-term survival and could serve as a prognostic non-invasive marker.
RESUMO
Large-conductance Ca2+- and voltage-activated K+ (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene (Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca2+ binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K+, we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca2+ binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory ß and γ subunits. We finish this review by describing how the association of the α subunit with ß or with γ subunits can change the BK channel phenotype and pharmacology.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta , Animais , HumanosRESUMO
BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.
Assuntos
Nanopartículas , Linfócitos T , Humanos , Animais , Camundongos , Nanopartículas/química , Feminino , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Evasão da Resposta Imune , Neoplasias de Mama Triplo Negativas/terapia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous studies have suggested a negative impact of steroids on the efficacy of immune checkpoint inhibitors (ICI), but how this effect is modulated by the dosage and time of administration is yet to be clarified. We have performed a retrospective analysis of 475 patients with advanced solid tumors treated with ICI as monotherapy from 2015 to 2022. Data regarding immune-related adverse events (irAEs) and clinical outcomes were collected. For each patient, the daily steroid dose (in mg/kg of prednisone) was registered until disease progression or death. The impact of cumulative doses on response rates and survival outcomes was analyzed within different periods. The objective response rate (ORR) was significantly lower among patients exposed to steroids within 30 days before the first cycle of ICI (C1) (20.3% vs. 36.7%, p < 0.01) and within the first 90 days of treatment (25.7% vs. 37.7%, p = 0.01). This negative association was confirmed by multivariable analysis. Higher mean steroid doses were observed among non-responders, and cumulative doses were inversely correlated with the disease control rate (DCR) around ICI initiation. Remarkably, poorer outcomes were observed even in patients belonging to the lowest dose quartile compared to the steroid-naïve population. The exposure to steroids after 6 months of ICI was not associated with worse survival outcomes. Our results suggest that the potential impact of steroids on ICI efficacy may be time-dependent, prevailing around ICI initiation, and dose-dependent, with modulation of neutrophil-to-lymphocyte ratio as a possible underlying mechanism.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias , Humanos , Masculino , Feminino , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/mortalidade , Pessoa de Meia-Idade , Estudos Retrospectivos , Idoso , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/efeitos adversos , Imunoterapia/métodos , Adulto , Esteroides/uso terapêutico , Esteroides/administração & dosagem , Relação Dose-Resposta a Droga , Idoso de 80 Anos ou mais , Fatores de TempoRESUMO
Lignocellulosic material is a leading carbon source for economically viable biotechnological processes; however, compounds such furfural and acetic acid exhibit toxicity to yeasts. Nonetheless, research about the molecular mechanism of furfural and acetic acid toxicity is still scarce in yeasts like Scheffersomyces stipitis. Thus, this study aims to elucidate the impact of furfural and acetic acid on S. stipitis regarding bioenergetic and fermentation parameters. Here, we provide evidence that furfural and acetic acid induce a delay in cell growth and extend the lag phase. The mitochondrial membrane potential decreased in all treatments with no significant differences between inhibitors or concentrations. Interestingly, reactive oxygen species increased when the inhibitor concentrations were from 0.1 to 0.3 % (v/v). The glycolytic flux was not significantly (p > 0.05) altered by acetic acid, but furfural caused different effects. Ethanol production decreased significantly (4.32 g·L-1 in furfural and 5.06 g·L-1 in acetic acid) compared to the control (26.3 g·L-1). In contrast, biomass levels were not significantly different in most treatments compared to the control. This study enhances our understanding of the effects of furfural and acetic acid at the mitochondrial level in a pentose-fermenting yeast like S. stipitis.
RESUMO
IMPORTANCE: Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.