RESUMO
Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.
Assuntos
Bioimpressão/métodos , Reatores Biológicos , Hidrodinâmica , Modelos Biológicos , Perfusão/instrumentação , Animais , Linhagem Celular , Simulação por Computador , Desenho de Equipamento , Camundongos , Osteoblastos/citologiaRESUMO
BACKGROUND: Increased morbidity and mortality are well documented in Status 7(inactive list) patients. Delays in transplantation secondary to prolonged periods on inactive status also negatively impacts transplant outcomes. We developed an effective system to reduce the proportion of status 7 patients on our kidney transplant waitlist. This can easily be reproduced by other transplant centers since concerns about Status 7 list size are commonplace. METHODS: Meetings of a dedicated status 7 focus group were undertaken biweekly beginning in April 2016, each lasting for 1 hour or less. The group was led by a transplant physician and comprised of members from all disciplines of the kidney transplant department. Individual patient barriers to activation were systematically evaluated and action plans were developed to overcome those. The formal meetings were supplemented by updates to an electronic database accessible to all members of the team. RESULTS: In the first 2 years of the program, we were able to activate and eventually transplant 18% of the formerly inactive patients. Forty percent of all inactive patients were removed from the waitlist due to one or more unsurmountable barriers. The median time patients stayed inactive on the waitlist was shortened from 1344 days at the start of this initiative to 581 days at the end. CONCLUSION: This strategy of systematic reevaluation of status 7 patients resulted in successful disposition of a substantial number of inactive patients. Further, waitlist time was reduced and transplantation expedited for the appropriate individuals. This approach could easily be adapted by other transplant centers with minimum utilization of resources.
Assuntos
Grupos Focais , Transplante de Rim/estatística & dados numéricos , Desenvolvimento de Programas/estatística & dados numéricos , Listas de Espera , Fatores Etários , Humanos , Pessoa de Meia-Idade , Fatores de Tempo , Tempo para o Tratamento/estatística & dados numéricos , Listas de Espera/mortalidadeRESUMO
Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERß, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.
Assuntos
Ciclina D1/biossíntese , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/biossíntese , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Benzofuranos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/genética , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Indóis/administração & dosagem , Panobinostat , Receptor ErbB-2/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: The purpose of this study was to systematically review and meta-analyze dynamic contrast-enhanced MRI (DCE-MRI) for the detection of prostate cancer in comparison with standard evaluation with T2-weighted imaging. MATERIALS AND METHODS: A PubMed electronic database search for the terms "dynamic contrast-enhanced," "prostate," and "MRI" was completed for articles up to September 17, 2013. All included studies had histopathologic correlation. Two by two contingency data were constructed for each study. A binormal bayesian ROC model was used to estimate and compare sensitivity, specificity, and AUC among eligible modalities. RESULTS: Both DCE-MRI (0.82-0.86) and diffusion-weighted MRI (DWI) (0.84-0.88) yielded significantly better AUC than T2-weighted imaging (0.68-0.77). Moreover, partial AUC for the combination of DCE-MRI, DWI, and T2-weighted imaging was improved significantly (0.111; 0.103-0.119) when compared with DCE-MRI alone (0.079; 0.072-0.085) and T2-weighted imaging alone (0.079; 0.074-0.084) but not DWI alone (0.099; 0.091-0.108). Sensitivity and specificity were similar among the four modalities. CONCLUSION: DCE-MRI improves AUC of tumor detection overall compared with T2-weighted imaging alone. Methods for DCE-MRI analysis require standardization, but visual analysis performs similar to semiquantitative methods. A two-parameter approach using DCE-MRI and T2-weighted imaging or DWI and T2-weighted imaging may be sufficient, and the latter may be more favorable for most routine prostate cancer imaging.
Assuntos
Meios de Contraste , Imageamento por Ressonância Magnética/métodos , Neoplasias da Próstata/diagnóstico , Imagem de Difusão por Ressonância Magnética , Humanos , Masculino , Neoplasias da Próstata/patologia , Sensibilidade e EspecificidadeRESUMO
This article describes a Council on Social Work Education (CSWE) Gero-Ed Center BEL Project's activities and reports its final outcomes. An oral history interview in paired human behavior and practice skills courses addressed gerontological social work competencies focused on assessing and addressing values and biases regarding aging, and the ability to relate concepts and theories of aging to practice. Significant increases in perceived proficiency in these competencies occurred, as did significant decreases in negative attitudes toward older people and working with older adults. Qualitative data supported these results. Findings suggest social work educators utilize a combination of classroom-based and experiential learning to maximize student development.
Assuntos
Etarismo/prevenção & controle , Geriatria/educação , Dinâmica Populacional/tendências , Aprendizagem Baseada em Problemas/métodos , Serviço Social/educação , Adulto , Idoso , Etarismo/psicologia , Envelhecimento , Geriatria/métodos , Geriatria/normas , Humanos , Relação entre Gerações , Entrevistas como Assunto/métodos , Masculino , Pessoa de Meia-Idade , Avaliação de Programas e Projetos de Saúde/métodos , Pesquisa Qualitativa , Autoavaliação (Psicologia) , Serviço Social/métodos , Serviço Social/normas , Estudantes/psicologia , Adulto JovemRESUMO
Translational research is a continuum between clinical and basic research where the patient is the center of the research process. It brings clinical research to a starting point for the drug discovery process, permitting the generation of a more robust pathophysiological hypothesis essential for a better selection of drug targets and candidate optimization. It also establishes the basis of early proof for clinical concept studies, preferably in phase I, for which biomarkers and surrogate endpoints can often be used. Systems biology is a prerequisite approach to translational research where technologies and expertise are integrated and articulated to support efficient and productive realization of this concept. The first component of systems biology relies on omics-based technologies and integrates the changes in variables, such as genes, proteins and metabolites, into networks that are responsible for an organism's normal and diseased state. The second component of systems biology is in the domain of computational methods, where simulation and modeling create hypotheses of signaling pathways, transcription networks, physiological processes or even cell- or organism-based models. The simulations aim to show the origin of perturbations of the system that lead to pathological states and what treatment could be achieved to ameliorate or normalize the system. This review discusses how translational research and systems biology together could improve global understanding of drug targets, suggest new targets and approaches for therapeutics, and provide a deeper understanding of drug effects. Taken together, these types of analyses can lead to new therapeutic options while improving the safety and efficacy of new and existing medications.
Assuntos
Descoberta de Drogas/métodos , Biologia de Sistemas/métodos , Animais , Humanos , Pesquisa Translacional BiomédicaRESUMO
INTRODUCTION: Clinical markers of response in metastatic renal cell carcinoma (mRCC) are lacking. Low hemoglobin (Hb) is associated with poor outcomes in the IMDC risk score. This study evaluates the role of Hb as a marker of treatment outcomes in mRCC. PATIENTS AND METHODS: This multicenter retrospective study evaluated 276 patients with mRCC treated with frontline immune checkpoint inhibitor (ICI) therapy, ICI and vascular endothelial growth factor (VEGF) inhibitor (VEGFI) combinations (ICI/VEGFI), or VEGFI monotherapy between 2014 and 2021. Hb levels at baseline, week 6 and 12 and at disease progression or death were recorded. Patients were categorized as responders (CR+PR) or nonresponders (SD+PD) using cross-sectional imaging at week 12. The association between baseline and dynamic changes in Hb and oncological outcomes was assessed. RESULTS: Thirty-seven percent, 40% and 22% of patients received ICIs, ICI/VEGFI and VEGFI respectively. In patients receiving ICIs, there was a significant increase in Hb amongst responders from baseline to week 12 (P= .02). Amongst patients receiving ICI/VEGFI, there was an increase in Hb from baseline to week 12 which was greater in responders (P< .001). In patients receiving VEGFI monotherapy, responders had a higher Hb at baseline (P= .01), week 6 (P= .04), and week 12 (P= .003). An increase in Hb was a significant independent predictor of progression-free survival amongst patients receiving ICIs (HR 0.40, 95%CI, 0.19-0.83, P= .009). CONCLUSION: Baseline and dynamic changes in Hb are associated with first-line treatment outcomes in patients with mRCC and represent a pragmatic early serological marker.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Estudos Retrospectivos , Fator A de Crescimento do Endotélio Vascular , Inibidores de Proteínas Quinases/uso terapêutico , Prognóstico , Inibidores da Angiogênese/uso terapêutico , HemoglobinasRESUMO
Axial flow left ventricular assist devices (LVADs) are a significant improvement in mechanical circulatory support. However, patients with these devices experience degradation of large von Willebrand factor (vWF) multimers, which is associated with bleeding and may be caused by high shear stresses within the LVAD. In this study, we used computational fluid mechanics to determine the wall shear stresses, shear rates, and residence times in a centrifugal LVAD and assess the impact on these variables caused by changing impeller speed and changing from a shrouded to a semi-open impeller. In both LVAD types, shear rates were well over 10,000/s in several regions. This is high enough to degrade vWF, but it is unclear if residence times, which were below 5ms in high-shear regions, are long enough to allow vWF cleavage. Additionally, wall shear stresses were below the threshold stress of 10Pa only in the outlet tube so it is feasible to endothelialize this region to enhance its biocompatibility.
Assuntos
Coração Auxiliar , Hemodinâmica , Hidrodinâmica , Desenho de Equipamento , Humanos , Estresse Mecânico , Função Ventricular , Fator de von Willebrand/metabolismoRESUMO
Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.
Assuntos
Antibacterianos/farmacologia , Daptomicina/análogos & derivados , Animais , Antibacterianos/biossíntese , Antibacterianos/química , Bovinos , Daptomicina/biossíntese , Daptomicina/química , Daptomicina/farmacologia , Feminino , Genes Bacterianos , Bactérias Gram-Positivas/efeitos dos fármacos , Lipoproteínas/biossíntese , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pneumonia Pneumocócica/tratamento farmacológico , Engenharia de Proteínas , Surfactantes Pulmonares/administração & dosagem , Staphylococcus aureus/efeitos dos fármacos , Streptomyces/genéticaRESUMO
A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu12), at the expense of factors containing 3-methyl-glutamic acid (3mGlu12). This provided a practical route to produce high levels of pure Glu12-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.
Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Produtos Biológicos/biossíntese , Produtos Biológicos/genética , Cromossomos Artificiais Bacterianos , Conjugação Genética , Escherichia coli/genética , Teste de Complementação Genética , Engenharia Genética/métodos , Vetores Genéticos , Genética Microbiana/métodos , Lipoproteínas/biossíntese , Lipoproteínas/genética , Plasmídeos , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.
RESUMO
Optogenetic stimulation of inhibitory interneurons has become a commonly used strategy for silencing neuronal activity. This is typically achieved using transgenic mice expressing excitatory opsins in inhibitory interneurons throughout the brain, raising the question of how spatially extensive the resulting inhibition is. Here, we characterize neuronal silencing in VGAT-ChR2 mice, which express channelrhodopsin-2 in inhibitory interneurons, as a function of light intensity and distance from the light source in several cortical and subcortical regions. We show that light stimulation, even at relatively low intensities, causes inhibition not only in brain regions targeted for silencing but also in their subjacent areas. In contrast, virus-mediated expression of an inhibitory opsin enables robust silencing that is restricted to the region of opsin expression. Our results reveal important constraints on using inhibitory interneuron activation to silence neuronal activity and emphasize the necessity of carefully controlling light stimulation parameters when using this silencing strategy.
Assuntos
Channelrhodopsins/metabolismo , Inativação Gênica , Interneurônios/metabolismo , Inibição Neural , Optogenética , Animais , Hipocampo/metabolismo , Luz , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Opsinas/metabolismo , Córtex Somatossensorial/metabolismo , Tálamo/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
BACKGROUND AND AIMS: The transcription factors FOXF1 and FOXF2 have been implicated in the development of the gastrointestinal tract but their role in adults or in gastrointestinal diseases is poorly understood. We have recently shown that expression of serum response factor (SRF), a transcription factor whose activity is modulated by FOXF proteins, is decreased in the stomach muscularis of patients with gastroparesis. The aim of the current study was to determine whether FOXF expression is decreased in gastroparesis patients and whether loss of FOXF1 and/or FOXF2 from adult smooth muscle is sufficient to impair gastric emptying in mice. METHODS: Full-thickness stomach biopsy samples were collected from control subjects and from patients with gastroparesis. mRNA was isolated from the muscularis externa, and FOXF mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. Foxf1 and Foxf2 were knocked out together and separately from smooth muscle cells in adult mice, and the subsequent effect on liquid gastric emptying and contractile protein expression was determined. KEY RESULTS: Expression of FOXF1 and FOXF2 is decreased in smooth muscle tissue from gastroparesis patients. Knockout of Foxf1 and Foxf2 together, but not alone, from mouse smooth muscle resulted in delayed liquid gastric emptying. Foxf1/2 double knockout mice had decreased expression of smooth muscle contractile proteins, SRF, and myocardin in stomach muscularis. CONCLUSIONS AND INFERENCES: Our findings suggest that decreased expression of FOXF1 and FOXF2 may be contributing to the impaired gastric emptying seen in gastroparesis patients.
Assuntos
Fatores de Transcrição Forkhead/genética , Gastroparesia/genética , Adulto , Animais , Biópsia , Complicações do Diabetes/genética , Feminino , Esvaziamento Gástrico , Gastroparesia/patologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Estômago/patologiaRESUMO
The complement (C) system plays an important role in myelin breakdown during Wallerian degeneration (WD). The pathway and mechanism involved are, however, not clear. In a crush injury model of the sciatic nerve, we show that C6, necessary for the assembly of the membrane attack complex (MAC), is essential for rapid WD. At 3 d after injury, pronounced WD occurred in wild-type animals, whereas the axons and myelin of C6-deficient animals appeared intact. Macrophage recruitment and activation was inhibited in C6-deficient rats. However, 7 d after injury, the distal part of the C6-deficient nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wild-type nerves. Reconstitution of the C6-deficient animals with C6 restored the wild-type phenotype. Treatment with rhC1INH (recombinant human complement 1 inhibitor) blocked deposition of activated C-cleaved products after injury. These experiments demonstrate that the classical pathway of the complement system is activated after acute nerve trauma and that the entire complement cascade, including MAC deposition, is essential for rapid WD and efficient clearance of myelin after acute peripheral nerve trauma.
Assuntos
Complemento C6/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/metabolismo , Proteínas Inativadoras do Complemento 1/administração & dosagem , Proteína Inibidora do Complemento C1 , Complemento C6/administração & dosagem , Complemento C6/deficiência , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica/métodos , Antígeno Ki-1/metabolismo , Macrófagos/metabolismo , Masculino , Proteínas de Neurofilamentos/metabolismo , Ratos , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Neuropatia Ciática/complicações , Neuropatia Ciática/patologia , Serpinas/administração & dosagem , Degeneração Walleriana/etiologia , Degeneração Walleriana/genéticaRESUMO
Daptomycin is a lipopeptide antibiotic produced by a nonribosomal peptide synthetase (NRPS) in Streptomyces roseosporus. The holoenzyme is composed of three subunits, encoded by the dptA, dptBC, and dptD genes, each responsible for incorporating particular amino acids into the peptide. We introduced expression plasmids carrying dptD or NRPS genes encoding subunits from two related lipopeptide biosynthetic pathways into a daptomycin nonproducing strain of S. roseosporus harboring a deletion of dptD. All constructs successfully complemented the deletion in trans, generating three peptide cores related to daptomycin. When these were coupled with incomplete methylation of 1 amino acid and natural variation in the lipid side chain, 18 lipopeptides were generated. Substantial amounts of nine of these compounds were readily obtained by fermentation, and all displayed antibacterial activity against gram-positive pathogens.
Assuntos
Antibacterianos/farmacologia , Engenharia Genética , Lipoproteínas/farmacologia , Peptídeo Sintases/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Antibacterianos/biossíntese , Sequência de Bases , Clonagem Molecular , Daptomicina/química , Daptomicina/farmacologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Lipoproteínas/biossíntese , Lipoproteínas/química , Metilação , Dados de Sequência Molecular , Família Multigênica , Peptídeo Sintases/química , Peptídeo Sintases/genética , Plasmídeos , Subunidades Proteicas , Streptomyces/genéticaRESUMO
The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P<0.001) AMPA+S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.
Assuntos
Benzotiadiazinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Transtornos Mentais/tratamento farmacológico , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases , RNA Mensageiro/metabolismo , Ratos , Receptores de AMPA/metabolismoRESUMO
Sepsis can induce an overwhelming systemic inflammatory response, resulting in organ damage and death. Suppressor of cytokine signaling 1 (SOCS1) negatively regulates signaling by cytokine receptors and Toll-like receptors (TLRs). However, the cellular targets and molecular mechanisms for SOCS1 activity during polymicrobial sepsis are unknown. To address this, we utilized a cecal ligation and puncture (CLP) model for sepsis; C57BL/6 mice subjected to CLP were then treated with a peptide (iKIR) that binds the SOCS1 kinase inhibitory region (KIR) and blocks its activity. Treatment with iKIR increased CLP-induced mortality, bacterial burden, and inflammatory cytokine production. Myeloid cell-specific SOCS1 deletion (Socs1Δmyel) mice were also more susceptible to sepsis, demonstrating increased mortality, higher bacterial loads, and elevated inflammatory cytokines, compared with Socs1fl littermate controls. These effects were accompanied by macrophage metabolic reprograming, as evidenced by increased lactic acid production and elevated expression of the glycolytic enzymes hexokinase, lactate dehydrogenase A, and glucose transporter 1 in septic Socs1Δmyel mice. Upregulation was dependent on the STAT3/HIF-1α/glycolysis axis, and blocking glycolysis ameliorated increased susceptibility to sepsis in iKIR-treated CLP mice. These results reveal a role of SOCS1 as a regulator of metabolic reprograming that prevents overwhelming inflammatory response and organ damage during sepsis.
RESUMO
BACKGROUND: The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation. METHODS: We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined. RESULTS: Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively). CONCLUSION: These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.
RESUMO
A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.
Assuntos
Antibacterianos/biossíntese , Streptomyces/metabolismo , Antibacterianos/química , Asparagina/análogos & derivados , Asparagina/biossíntese , Asparagina/química , Asparagina/genética , Lipoproteínas/biossíntese , Lipoproteínas/química , Lipoproteínas/genética , Espectroscopia de Ressonância Magnética , Rotação Ocular , Sarcosina/análogos & derivados , Sarcosina/biossíntese , Sarcosina/química , Sarcosina/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Streptomyces/genéticaRESUMO
A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.