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1.
RNA ; 30(9): 1213-1226, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-38918043

RESUMO

Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.


Assuntos
Inibidores Enzimáticos , Quadruplex G , Telomerase , Telômero , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telomerase/genética , Humanos , Telômero/metabolismo , Quadruplex G/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Ácidos Picolínicos/farmacologia , Ácidos Picolínicos/química , Replicação do DNA/efeitos dos fármacos , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/metabolismo , DNA/metabolismo , Aminoquinolinas , Porfirinas , DNA Primase
2.
Genes Dev ; 32(21-22): 1380-1397, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366907

RESUMO

Cells undergo metabolic adaptation during environmental changes by using evolutionarily conserved stress response programs. This metabolic homeostasis is exquisitely regulated, and its imbalance could underlie human pathological conditions. We report here that C9orf72, which is linked to the most common forms of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a key regulator of lipid metabolism under stress. Loss of C9orf72 leads to an overactivation of starvation-induced lipid metabolism that is mediated by dysregulated autophagic digestion of lipids and increased de novo fatty acid synthesis. C9orf72 acts by promoting the lysosomal degradation of coactivator-associated arginine methyltransferase 1 (CARM1), which in turn regulates autophagy-lysosomal functions and lipid metabolism. In ALS/FTD patient-derived neurons or tissues, a reduction in C9orf72 function is associated with dysregulation in the levels of CARM1, fatty acids, and NADPH oxidase NOX2. These results reveal a C9orf72-CARM1 axis in the control of stress-induced lipid metabolism and implicates epigenetic dysregulation in relevant human diseases.


Assuntos
Proteína C9orf72/fisiologia , Glucose/fisiologia , Metabolismo dos Lipídeos , Proteína-Arginina N-Metiltransferases/metabolismo , Estresse Fisiológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Cultivadas , Ácidos Graxos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteína-Arginina N-Metiltransferases/fisiologia
3.
Infect Immun ; 92(5): e0000624, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38629806

RESUMO

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.


Assuntos
Receptor 1 de Quimiocina CX3C , Colo , Enterococcus faecalis , Macrófagos , Receptores CCR2 , Receptores de Quimiocinas , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Macrófagos/microbiologia , Macrófagos/imunologia , Camundongos , Colo/microbiologia , Colo/imunologia , Receptores CCR2/metabolismo , Receptores CCR2/genética , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/genética , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Camundongos Endogâmicos C57BL , Linfonodos/microbiologia , Linfonodos/imunologia , Receptores CCR7/metabolismo , Receptores CCR7/genética
4.
J Biol Chem ; 294(26): 10290-10299, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31110048

RESUMO

CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Clivagem do DNA , Sistema Imunitário , RNA/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Pareamento de Bases , Proteínas Associadas a CRISPR/genética , Plasmídeos/genética , RNA/genética
5.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728267

RESUMO

Gammaherpesviruses are ubiquitous pathogens that are associated with B cell lymphomas. In the early stages of chronic infection, these viruses infect naive B cells and subsequently usurp the B cell differentiation process through the germinal center response to ensure latent infection of long-lived memory B cells. A unique feature of early gammaherpesvirus chronic infection is a robust differentiation of irrelevant, virus-nonspecific B cells with reactivities against self-antigens and antigens of other species. In contrast, protective, virus-specific humoral responses do not reach peak levels until a much later time. While several host factors are known to either promote or selectively restrict gammaherpesvirus-driven germinal center response, viral mechanisms that contribute to the irrelevant B cell response have not been defined. In this report we show that the expression and the enzymatic activity of the gammaherpesvirus-encoded conserved protein kinase selectively facilitates the irrelevant, but not virus-specific, B cell responses. Further, we show that lack of interleukin-1 (IL-1) receptor attenuates gammaherpesvirus-driven B cell differentiation and viral reactivation. Because germinal center B cells are thought to be the target of malignant transformation during gammaherpesvirus-driven lymphomagenesis, identification of host and viral factors that promote germinal center responses during gammaherpesvirus infection may offer an insight into the mechanism of gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous cancer-associated pathogens that usurp the B cell differentiation process to establish life-long latent infection in memory B cells. A unique feature of early gammaherpesvirus infection is the robust increase in differentiation of B cells that are not specific for viral antigens and instead encode antibodies that react with self-antigens and antigens of other species. Viral mechanisms that are involved in driving such irrelevant B cell differentiation are not known. Here, we show that gammaherpesvirus-encoded conserved protein kinase and host IL-1 signaling promote irrelevant B cell responses and gammaherpesvirus-driven germinal center responses, with the latter thought to be the target of viral transformation.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Gammaherpesvirinae/imunologia , Ativação Linfocitária , Proteínas Quinases/imunologia , Proteínas Virais/imunologia , Animais , Linfócitos B/patologia , Gammaherpesvirinae/genética , Centro Germinativo/imunologia , Centro Germinativo/patologia , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Camundongos , Camundongos Knockout , Proteínas Quinases/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Proteínas Virais/genética
6.
J Surg Res ; 247: 524-529, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31668431

RESUMO

BACKGROUND: The appropriate duration of antibiotic therapy for surgical site infection (SSI) prevention in traumatic mandibular fracture repair is unknown, and practices vary significantly. The objective of this study was to characterize antibiotic duration and outcomes after surgical repair of traumatic mandibular fracture. METHODS: A single-center, retrospective analysis of all adult patients who underwent surgical fixation of a mandible fracture between January 2014 and December 2016 was performed. Operative service was categorized between otolaryngology (ear, nose, and throat surgery), plastic and reconstructive surgery, and oral and maxillofacial services. Primary outcomes were SSI and operative complications (including osteomyelitis, nonunion, malocclusion, and hardware infections). Differences in antibiotic prescription pattern were analyzed using analysis of variance test and Pearson chi-squared test. RESULTS: A total of 75 patients were included in the study with 33 (44.0%), 26 (34.7%), and 16 (21.3%) managed by plastic and reconstructive surgery, ear, nose, and throat surgery, and oral and maxillofacial services, respectively. Median age was 30.0 y. Median injury severity score was 4.0. There was no significant difference in hospital length of stay (P = 0.44), intensive care unit length of stay (P = 0.53), or postoperative complications (P = 0.15). None of our patients developed an SSI or postantibiotics complications. Although the total inpatient duration of antibiotics was not significantly different among services (P = 0.37), there were significant differences in outpatient duration of antibiotics (P = 0.007) and total duration of antibiotics (P = 0.003). CONCLUSIONS: Duration of antibiotics is not associated with postoperative SSI or antibiotics-related complications. The wide variation in prescribing practices and lack of any clear benefit for prolonged antibiotics provides an opportunity to explore the benefits of a standardized short course of antibiotics. LEVEL OF EVIDENCE: Therapeutic study, III.


Assuntos
Antibacterianos/administração & dosagem , Antibioticoprofilaxia/métodos , Fixação de Fratura/efeitos adversos , Fraturas Mandibulares/cirurgia , Traumatismos Mandibulares/complicações , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Antibioticoprofilaxia/normas , Antibioticoprofilaxia/estatística & dados numéricos , Esquema de Medicação , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Mandíbula/cirurgia , Fraturas Mandibulares/etiologia , Traumatismos Mandibulares/diagnóstico , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Sistema de Registros/estatística & dados numéricos , Estudos Retrospectivos , Infecção da Ferida Cirúrgica/etiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
7.
Soc Sci Res ; 87: 102398, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32279857

RESUMO

Women entered the paid workforce in unprecedented numbers during the 20th century. Yet recent years have been witness to a creeping reversal in women's labor force participation. Why did the revolution stall? In response to debates over a "natural" limit to women's employment, or a cultural backlash against the dual-breadwinner household, we consider an alternative explanation, namely whether immigration has slowed the growth in female labor force participation. Using CPS data from 1998 to 2018, we show that the increase in the share of immigrants and children of immigrants in the population has reduced overall female labor force participation. However, immigration accounts for relatively little of the retreat from the labor force. Instead, the compositional effect of population change is overshadowed by behavioral shifts that affect both natives and immigrants. Lower participation rates among native-born women accounts for most of the overall decline. Despite persistent differences, we also find substantial convergence in the labor force behavior of native-born and foreign-born women, which bodes well for the long-term economic incorporation of immigrants and their children.

8.
Langmuir ; 34(25): 7488-7496, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29874091

RESUMO

We study bacteriorhodopsin (BR) in its native purple membrane encapsulated within amorphous titanium dioxide, or titania, gels and in the presence of titania sol particles to explore this system for hydrogen production. Förster resonance energy transfer between BR and titanium dioxide sol particles was used to conclude that there is nanometer-scale proximity of bacteriorhodopsin to the titanium dioxide. The detection of BR-titania sol aggregates by fluorescence anisotropy and particle sizing indicated the affinity amorphous titania has for BR without the use of additional cross-linkers. UV-vis spectroscopy of BR-titania gels shows that methanol addition did not denature BR at a 25 mM concentration presence as a sacrificial electron donor. Additionally, confinement of BR in the gels significantly limited protein denaturation at higher concentration of added methanol or ethanol. Subsequently, titania gels fabricated through the sol-gel process using a titanium ethoxide precursor, water, and the addition of 25 mM methanol were used to encapsulate BR and a platinum reduction catalyst for the production of hydrogen gas under white light irradiation. The inclusion of 5 µM bacteriorhodopsin resulted in a hydrogen production rate of about 3.8 µmol hydrogen mL-1 h-1, an increase of 52% compared to gels containing no protein. Electron transfer and proton pumping by BR in close proximity to the titania gel surface are feasible explanations for the enhanced production of hydrogen without the need to cross-link BR to the titania gel. This work sets the stage for further developments of amorphous, rather than crystalline, titania-encapsulated bacteriorhodopsin for solar-driven hydrogen production through water splitting.

9.
Nature ; 548(7669): 527-528, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28783729
10.
PLoS Pathog ; 11(3): e1004705, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25756944

RESUMO

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹â»³8 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹â»³8 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.


Assuntos
Proteínas de Bactérias/imunologia , Infecções por Enterobacteriaceae/imunologia , Interações Hospedeiro-Parasita/fisiologia , Metaloproteases/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas de Bactérias/metabolismo , Citrobacter rodentium , Infecções por Enterobacteriaceae/metabolismo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Inflamação/imunologia , Inflamação/metabolismo , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/imunologia , Proteínas Ribossômicas/metabolismo , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Transfecção
11.
Environ Toxicol ; 32(5): 1513-1529, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27696670

RESUMO

Several estuaries along the Pacific Ocean coast of North America were identified recently as having elevated 4-nonylphenol (4-NP) in sediments and biota, raising concerns about reproductive impacts for wildlife given 4-NP's established estrogenic activity as an endocrine-disrupting compound. Here we characterize 4-NP mediated induction and recovery of estrogen-sensitive gene transcripts in the arrow goby (Clevelandia ios), an intertidal fish abundant in estuarine mud flats on the west coast of North America. Male gobies were exposed to waterborne 4-NP at 10 µg/L or 100 µg/L for 20 days followed by a 20 day depuration period. Additional males were treated with 17ß-estradiol (E2; 50 ng/L). 4-NP at 100 µg/L elevated hepatic mRNAs encoding vitellogenins A (vtgA) and C (vtgC) and choriogenin L (chgL) within 72 h, and choriogenin H minor (chgHm) within 12 days. Hepatic mRNAs encoding estrogen receptor alpha (esr1) were also elevated after 12 days of 4-NP exposure, but returned to pre-exposure levels at 20 days even under continuing 4-NP treatment. 4-NP did not alter mRNA levels of estrogen receptor gamma (esr2a) in the liver, or of esr1, esr2a, and cytochrome P450 aromatase B (cyp19a1b) in the brain. The temporal pattern of initial induction for hepatic vtgA, vtgC, and chgL transcripts by 4-NP mirrored the pattern by E2, while chgHm and esr1 mRNA induction by 4-NP lagged 2-11 days behind the responses of these transcripts to E2. These findings establish 4-NP concentration- and time-dependent induction patterns of choriogenin and vitellogenin transcription following exposure to environmentally relevant 4-NP concentrations, while concurrently demonstrating tissue-specific induction patterns for esr1 by estrogenic compounds. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1513-1529, 2017.


Assuntos
Disruptores Endócrinos/farmacologia , Estradiol/farmacologia , Peixes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fenóis/farmacologia , Poluentes Químicos da Água/farmacologia , Animais , Biomarcadores/metabolismo , Estuários , Feminino , Peixes/genética , Peixes/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Fatores de Tempo , Vitelogeninas/genética
12.
ACS Omega ; 8(32): 29789-29793, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37599948

RESUMO

In nuclear reactors that use molten fluoride salts, either as coolants or as the medium for the fuel, the purity of the salts is critical for controlling salt chemistry and mitigating corrosion. Water is a particularly important contaminant in this regard, as it participates in a number of important corrosion reactions, so the careful measurement of oxygen, which is principally present in the salts due to water contamination, is a critical step in salt characterization. Here, we present an analytical method for quantifying oxygen contamination in Li2BeF4 (FLiBe), a technologically important and suitably representative fluoride salt, with a detection limit of 22 µg of oxygen, or 110 ppm in a 200 mg sample. To test the method, four FLiBe samples from different batches were tested. Two of these showed oxygen concentrations below the method detection limit, while two showed concentrations above it. In particular, the difference in the oxygen concentration between purified and un-purified batches of material from Kairos Power showed the efficacy of this method in characterizing the degree of oxygen removal obtained from purification methods.

13.
Neurotrauma Rep ; 4(1): 464-477, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37528868

RESUMO

Apolipoprotein E epsilon 4 (ApoE4) is the second most common variant of ApoE, being present in ∼14% of the population. Clinical reports identify ApoE4 as a genetic risk factor for poor outcomes after traumatic spinal cord injury (SCI) and spinal cord diseases such as cervical myelopathy. To date, there is no intervention to promote recovery of function after SCI/spinal cord diseases that is specifically targeted at ApoE4-associated impairment. Studies in the human and mouse brain link ApoE4 to elevated levels of synaptojanin 1 (synj1), a lipid phosphatase that degrades phosphoinositol 4,5-bisphosphate (PIP2) into inositol 4-monophosphate. Synj1 regulates rearrangements of the cytoskeleton as well as endocytosis and trafficking of synaptic vesicles. We report here that, as compared to ApoE3 mice, levels of synj1 messenger RNA and protein were elevated in spinal cords of healthy ApoE4 mice associated with lower PIP2 levels. Using a moderate-severity model of contusion SCI in mice, we found that genetic reduction of synj1 improved locomotor function recovery at 14 days after SCI in ApoE4 mice without altering spared white matter. Genetic reduction of synj1 did not alter locomotor recovery of ApoE3 mice after SCI. Bulk RNA sequencing revealed that at 14 days after SCI in ApoE4 mice, genetic reduction of synj1 upregulated genes involved in glutaminergic synaptic transmission just above and below the lesion. Overall, our findings provide evidence for a link between synj1 to poor outcomes after SCI in ApoE4 mice, up to 14 days post-injury, through mechanisms that may involve the function of excitatory glutaminergic neurons.

14.
Front Cell Neurosci ; 17: 1163436, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37416508

RESUMO

Membrane channels such as those formed by connexins (Cx) and P2X7 receptors (P2X7R) are permeable to calcium ions and other small molecules such as adenosine triphosphate (ATP) and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx and Panx1 hemichannels (HCs). To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X7R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in spinal cord tissue just caudal from the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

15.
bioRxiv ; 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36824813

RESUMO

Membrane channels such as connexins (Cx), pannexins (Panx) and P2X 7 receptors (P2X 7 R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X 7 R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

16.
J Exp Med ; 220(1)2023 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-36315049

RESUMO

Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.


Assuntos
Linfócitos T CD8-Positivos , Infecção Persistente , Humanos , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Diferenciação Celular , Receptores de Antígenos de Linfócitos T/metabolismo
17.
Front Mol Neurosci ; 16: 1183315, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37692100

RESUMO

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

18.
Gen Comp Endocrinol ; 172(3): 505-17, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21549118

RESUMO

In fish as in other vertebrates, the diverse functions of thyroid hormones are mediated at the peripheral tissue level through iodothyronine deiodinase (dio) enzymes and thyroid hormone receptor (tr) proteins. In this study, we examined thyroid hormone regulation of mRNAs encoding the three deiodinases dio1, dio2 and dio3 - as well as three thyroid hormone receptors trαA, trαB and trß - in initial phase striped parrotfish (Scarus iseri). Parrotfish were treated with dissolved phase T(3) (20 nM) or methimazole (3 mM) for 3 days. Treatment with exogenous T(3) elevated circulating T(3), while the methimazole treatment depressed plasma T(4). Experimentally-induced hyperthyroidism increased the relative abundance of transcripts encoding trαA and trß in the liver and brain, but did not affect trαB mRNA levels in either tissue. In both sexes, methimazole-treated fish exhibited elevated dio2 transcripts in the liver and brain, suggesting enhanced outer-ring deiodination activity in these tissues. Accordingly, systemic hyperthyroidism elevated relative dio3 transcript levels in these same tissues. In the gonad, however, patterns of transcript regulation were distinctly different with elevated T(3) increasing mRNAs encoding dio2 in testicular and ovarian tissues and dio3, trαA and trαB in the testes only. Thyroid hormone status did not affect dio1 transcript abundance in the liver, brain or gonads. Taken as a whole, these results demonstrate that thyroidal status influences relative transcript abundance for dio2 and dio3 in the liver, provide new evidence for similar patterns of dio2 and dio3 mRNA regulation in the brain, and make evident that fish exhibit tr subtype-specific transcript abundance changes to altered thyroid status.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica , Iodeto Peroxidase/genética , Perciformes/genética , RNA Mensageiro/metabolismo , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/fisiologia , Animais , DNA Complementar/metabolismo , Proteínas de Peixes/metabolismo , Iodeto Peroxidase/metabolismo , Fígado/metabolismo , Metimazol/farmacologia , Perciformes/metabolismo , Filogenia , Receptores dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/sangue
19.
Front Vet Sci ; 8: 646437, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34277749

RESUMO

Background: Pimobendan provides a significant survival benefit in dogs with cardiac disease, including degenerative mitral valve disease and dilated cardiomyopathy (DCM). Its positive inotropic effect is well-known, however, it has complex effects and the mechanisms behind the survival benefit are not fully characterized. Secondary hemodynamic effects may decrease mitral regurgitation (MR) in DCM, and the benefits of pimobendan may extend to improved cardiac relaxation and improved atrial function. Hypothesis/Objectives: Our objective was to investigate the acute cardiac effects of pimobendan in dogs with a DCM phenotype. We hypothesized that pimobendan would increase left atrial (LA) contractility, reduce mitral regurgitation, improve diastolic function, and lower circulating NT-ProBNP levels. Animals: Seven purpose-bred Beagles were studied from a research colony with tachycardia induced DCM phenotype. Methods: The effects of pimobendan were studied under a placebo-controlled single-blinded cross-over design. In short, dogs underwent baseline and 3 h post-dose examinations 7 days apart with echocardiography and a blood draw. Dogs were randomized to receive oral placebo or 0.25 mg/kg pimobendan after their baseline exam. Investigators were blinded to treatments until all measurements were compiled. Results: When treated with pimobendan, the dogs had significant increases in systolic function and decreases in MR, compared to when treated with placebo. There were no detectable differences in left atrial measures, including LA size, LA emptying fraction, LA functional index or mitral A wave velocity. Heart rate decreased significantly with pimobendan compared to placebo. There was also a decrease in isovolumetric relaxation time normalized to heart rate. NT-proBNP levels had a high degree of variability. Conclusions: Improved mitral regurgitation severity and improved lusitropic function may contribute to the reported survival benefit for dogs with cardiac disease administered pimobendan. Pimobendan did not overtly improve LA function as assessed by echocardiography, and NT-proBNP was not significantly changed with a single dose of this medication. Further studies are needed to better characterize LA effects with other imaging modalities, to better quantify the total improvement of MR severity, and to assess chronic use of pimobendan on diastolic function in DCM.

20.
Mol Immunol ; 137: 94-104, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34242922

RESUMO

The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.


Assuntos
Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Animais , Proliferação de Células/fisiologia , Citocinas/imunologia , Feminino , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores de Células Matadoras Naturais/imunologia , Transdução de Sinais/imunologia , Transcriptoma/imunologia
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