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Reducing carbon dioxide (CO2) emissions urgently requires the large-scale deployment of carbon-capture technologies. These technologies must separate CO2 from various sources and deliver it to different sinks1,2. The quest for optimal solutions for specific source-sink pairs is a complex, multi-objective challenge involving multiple stakeholders and depends on social, economic and regional contexts. Currently, research follows a sequential approach: chemists focus on materials design3 and engineers on optimizing processes4,5, which are then operated at a scale that impacts the economy and the environment. Assessing these impacts, such as the greenhouse gas emissions over the plant's lifetime, is typically one of the final steps6. Here we introduce the PrISMa (Process-Informed design of tailor-made Sorbent Materials) platform, which integrates materials, process design, techno-economics and life-cycle assessment. We compare more than 60 case studies capturing CO2 from various sources in 5 global regions using different technologies. The platform simultaneously informs various stakeholders about the cost-effectiveness of technologies, process configurations and locations, reveals the molecular characteristics of the top-performing sorbents, and provides insights on environmental impacts, co-benefits and trade-offs. By uniting stakeholders at an early research stage, PrISMa accelerates carbon-capture technology development during this critical period as we aim for a net-zero world.
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Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.
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Antivirais/farmacologia , Proteínas de Repetição de Anquirina Projetadas/química , Temperatura , Sequência de Aminoácidos , Antivirais/química , Antivirais/uso terapêutico , COVID-19/virologia , Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , SARS-CoV-2/efeitos dos fármacos , Alinhamento de Sequência , Tratamento Farmacológico da COVID-19RESUMO
Adsorption is at the heart of many processes from gas separation to cooling. The design of adsorption-based processes requires equilibrium adsorption properties. However, data for adsorption equilibria are limited, and therefore, a model is desirable that uses as little data as possible for its parametrization, while allowing for data interpolation or even extrapolation. This work presents a physics-based model for adsorption isotherms and other equilibrium adsorption properties. The model is based on one-dimensional classical density functional theory (1D-DFT) and the perturbed-chain statistical associating fluid theory (PC-SAFT). The physical processes inside the pores are considered in a thermodynamically consistent approach that is computationally efficient. Once parametrized with a single isotherm, the model is able to extrapolate to other temperatures and outperforms the extrapolation capabilities of state-of-the-art models, such as the empirical isotherm models from Langmuir or Toth. Furthermore, standard combining rules can be used to transfer parameters adjusted to an adsorbent/fluid pair to other fluids. These features are demonstrated for the adsorption of N2, CH4, and CO2 in metal-organic frameworks. Thereby, the presented model can calculate temperature-dependent isotherms for various fluids by using data limited to a single isotherm as input.
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[This corrects the article DOI: 10.1039/D2DD00058J.].
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BACKGROUND: Acute appendicitis is a local intestinal inflammation with unclear origin. The aim was to test whether bacteria in appendicitis differ in composition to bacteria found in caecal biopsies from healthy and disease controls. METHODS AND PATIENTS: We investigated sections of 70 appendices using rRNA-based fluorescence in situ hybridisation. Four hundred caecal biopsies and 400 faecal samples from patients with inflammatory bowel disease and other conditions were used as controls. A set of 73 group-specific bacterial probes was applied for the study. RESULTS: The mucosal surface in catarrhal appendicitis showed characteristic lesions of single epithelial cells filled with a mixed bacterial population ('pinned cells') without ulceration of the surroundings. Bacteria deeply infiltrated the tissue in suppurative appendicitis. Fusobacteria (mainly Fusobacterium nucleatum and necrophorum) were a specific component of these epithelial and submucosal infiltrates in 62% of patients with proven appendicitis. The presence of Fusobacteria in mucosal lesions correlated positively with the severity of the appendicitis and was completely absent in caecal biopsies from healthy and disease controls. Main faecal microbiota represented by Bacteroides, Eubacterium rectale (Clostridium group XIVa), Faecalibacterium prausnitzii groups and Akkermansia muciniphila were inversely related to the severity of the disease. The occurrence of other bacterial groups within mucosal lesions of acute appendicitis was not related to the severity of the appendicitis. No Fusobacteria were found in rectal swabs of patients with acute appendicitis. CONCLUSIONS: Local infection with Fusobacterium nucleatum/necrophorum is responsible for the majority of cases of acute appendicitis.
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Apendicite/microbiologia , Infecções por Fusobacterium/complicações , Fusobacterium necrophorum/isolamento & purificação , Fusobacterium nucleatum/isolamento & purificação , Doença Aguda , Apendicectomia , Apendicite/patologia , Apendicite/cirurgia , Apêndice/microbiologia , Biópsia , Estudos de Casos e Controles , Ceco/microbiologia , Ceco/patologia , Fezes/microbiologia , Infecções por Fusobacterium/microbiologia , Humanos , Hibridização in Situ Fluorescente , Mucosa Intestinal/microbiologiaRESUMO
The knowledge of mixtures' phase equilibria is crucial in nature and technical chemistry. Phase equilibria calculations of mixtures require activity coefficients. However, experimental data on activity coefficients are often limited due to the high cost of experiments. For an accurate and efficient prediction of activity coefficients, machine learning approaches have been recently developed. However, current machine learning approaches still extrapolate poorly for activity coefficients of unknown molecules. In this work, we introduce a SMILES-to-properties-transformer (SPT), a natural language processing network, to predict binary limiting activity coefficients from SMILES codes. To overcome the limitations of available experimental data, we initially train our network on a large dataset of synthetic data sampled from COSMO-RS (10 million data points) and then fine-tune the model on experimental data (20 870 data points). This training strategy enables the SPT to accurately predict limiting activity coefficients even for unknown molecules, cutting the mean prediction error in half compared to state-of-the-art models for activity coefficient predictions such as COSMO-RS and UNIFACDortmund, and improving on recent machine learning approaches.
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Background: Malnutrition as well as overfeeding can have negative impacts on clinical outcomes in critically ill patients. Continuous veno-venous hemodialysis (CVVHD) with regional citrate anticoagulation (RCA) using trisodium citrate 4% (TSC) might play a role in nutrient disposition in patients in the ICU. Methods: In 33 consecutive patients on CVVHD with RCA, energy uptake or loss was calculated. Three macronutrients (lactate, glucose and citrate) were analyzed by taking prefilter blood and effluent samples. Results: Glucose and lactate clearance through CVVHD made up for a loss of 61 kcal/d (IQR 25−164 kcal/d) and 38 kcal/d (IQR 23−59 kcal/d), respectively. Two patients with hyperglycemic state (>350 mg/dL) lost around 600 kcal/d during CVVHD. Net post-filter citrate caloric delivery through RCA was 135 kcal/d (IQR: 124−144 kcal/d). Adding the three macronutrients, net caloric gain through CVVHD was 10 kcal/d (IQR: −63−75 kcal/d). Conclusion: In non-hyperglycemic patients on CVVHD with RCA, the metabolic contribution of the three macronutrients lactate, glucose and citrate is neglectable.
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Injúria Renal Aguda , Terapia de Substituição Renal Contínua , Humanos , Ácido Cítrico , Anticoagulantes/efeitos adversos , Glucose , Unidades de Terapia Intensiva , Lactatos , Injúria Renal Aguda/induzido quimicamenteRESUMO
Hypo- and hypernatremias are very frequent in intensive care unit (ICU) patients and are closely related to volume disturbances and volume management in the ICU. They are associated with longer ICU stays and significant increases in mortality. Treating them is more complex than it may initially appear. Hyponatremias are differentiated based on tonicity and volume status. With hypertonic and isotonic hyponatremias, the primary focus of treatment is the underlying hyperglycemia. In case of hypotonic hypovolemic hyponatremia, the condition is treated with balanced crystalloid solutions. In eu-/hypervolemic hypotonic hyponatremias acute treatment with hypertonic saline is necessary. Hypervolemic hypernatremia occurs almost exclusively in ICU patients, often due to infusion of hypertonic solutions. There is little evidence to guide treatment, although hypotonic infusions in conjunction with diuretics may represent a legitimate approach. Great emphasis should be placed on prevention and the infusion of hypertonic solutions should be avoided. Disturbances in plasma sodium concentrations are common, requiring close attention. Exact diagnostic classification needs to be made and volume managed accordingly.
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Hipernatremia , Hiponatremia , Desequilíbrio Hidroeletrolítico , Humanos , Hipernatremia/diagnóstico , Hipernatremia/terapia , Unidades de Terapia Intensiva , Soluções Isotônicas , Solução Salina Hipertônica/uso terapêutico , Desequilíbrio Hidroeletrolítico/diagnóstico , Desequilíbrio Hidroeletrolítico/terapiaRESUMO
Human bocavirus (HBoV) has to be considered a life-threatening pathogen in adults with atypical pneumonia. Pulsed high-dose glucocorticoid treatment may be beneficial in patients suffering from severe pulmonary disease caused by HBoV or other viruses. https://bit.ly/3epiMyO.
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OBJECTIVE: To study the incidence and distribution of adherent Gardnerella vaginalis. METHODS: Bacteria adherent to desquamated epithelial cells in the urine were detected using fluorescence in situ hybridization (FISH). Urine from patients with bacterial vaginosis (BV, n = 20), their partners (n = 10) and different control populations (n = 344) including pregnant women and their partners, randomly selected populations of hospitalized man, women and children as also healthy controls was investigated. RESULTS: Gardnerella was found in two different forms: cohesive and dispersed. In the cohesive form, Gardnerella were attached to the epithelial cells in groups of highly concentrated bacteria. In the dispersed form, solitary Gardnerella were intermixed with other bacterial groups. Cohesive Gardnerella was present in all patients with proven BV and their partners, in 7% of men and 13% of women hospitalized for reasons other than BV, in 16% of pregnant women and 12% of their male partners, and in none of the healthy laboratory staff or children. In sexual partners, occurrence of cohesive Gardnerella was clearly linked. Dispersed Gardnerella were found in 10-18% of randomly selected females, 3-4% of males and 10% of children and not sexually linked. In daily longitudinal investigations over 4 weeks no transition between cohesive and dispersed Gardnerella and vice versa was observed. Transmission of a cohesive Gardnerella strain could be followed retrospectively over 15 years using molecular genetic methods. CONCLUSIONS: Cohesive Gardnerella biofilm is a distinct, clearly definable entity which involves both genders and is sexually transmitted. The correct name distinguishing it from symptom-defined conditions like BV should be gardnerellosis and for the bacterium Gardnerella genitalis.
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Biofilmes , Gardnerella vaginalis/isolamento & purificação , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Adulto , Aderência Bacteriana , Bacteriúria/microbiologia , Criança , Pré-Escolar , Células Epiteliais/microbiologia , Feminino , Gardnerella vaginalis/genética , Genótipo , Hospitalização , Humanos , Hibridização in Situ Fluorescente , Masculino , Gravidez , Técnica de Amplificação ao Acaso de DNA Polimórfico , Estudos Retrospectivos , Parceiros Sexuais , Doenças Bacterianas Sexualmente Transmissíveis/transmissão , Urina/citologia , Urina/microbiologia , Vaginose Bacteriana/microbiologia , Vaginose Bacteriana/transmissão , Vaginose Bacteriana/urinaRESUMO
Recent data point at the similarity between the perianal and vaginal microflora in terms of Lactobacillus species involved. Bacterial vaginosis, the most common perturbation of the vaginal microflora involving primarily overgrowth of Gardnerella vaginalis, has also been suggested to involve a recto-vaginal pathway. We addressed this issue with regard to bacteria of the Bifidobacteriaceae family. In particular, we investigated the putative concordance of the presence of G. vaginalis and a series of Bifidobacteria between the perianal and vaginal microflora in 10 patients with bacterial vaginosis through multicolor fluorescence in situ hybridization analysis of desquamated epithelial cells. G. vaginalis was found in a biofilm mode of growth at the perianal and vaginal sites. In most women at least one of the following species was detected perianally: Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium breves, Bifidobacterium bifidum and Bifidobacterium catenulatum. At the vaginal site, none of these Bifidobacteria was found. We conclude that bacterial vaginosis does not occur as a result of simple growth per continuum of perianal bacteria. Only some species originating from the intestinal tract do display pronounced vaginotropism, like G. vaginalis, whereas many other species do not.
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Infecções por Bifidobacteriales/microbiologia , Bifidobacterium/isolamento & purificação , Gardnerella vaginalis/isolamento & purificação , Vagina/microbiologia , Vaginose Bacteriana/microbiologia , Bifidobacterium/genética , Biofilmes , Células Epiteliais/microbiologia , Feminino , Gardnerella vaginalis/genética , Humanos , Hibridização in Situ FluorescenteRESUMO
High mucosal and fecal concentrations of the antimicrobial siderophore-binding peptide Lipocalin-2 (Lcn2) are observed in inflammatory bowel disease. However, Lcn2 function in chronic intestinal inflammation remains unclear. Here, we demonstrate that Lcn2 protects from early-onset colitis and spontaneous emergence of right-sided colonic tumors resulting from IL-10 deficiency. Exacerbated inflammation in Lcn2(-/-)/Il10(-/-) mice is driven by IL-6, which also controls tumorigenesis. Lcn2(-/-)/Il10(-/-) mice exhibit profound alterations in gut microbial composition, which contributes to inflammation and tumorigenesis, as demonstrated by the transmissibility of the phenotype and protection conferred by antibiotics. Specifically, facultative pathogenic Alistipes spp. utilize enterobactin as iron source, bloom in Lcn2(-/-)/Il10(-/-) mice, and are sufficient to induce colitis and right-sided tumors when transferred into Il10(-/-) mice. Our results demonstrate that Lcn2 protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota.
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Colite/imunologia , Colite/microbiologia , Microbioma Gastrointestinal , Neoplasias Intestinais/imunologia , Neoplasias Intestinais/microbiologia , Lipocalina-2/imunologia , Animais , Bacteroides/crescimento & desenvolvimento , Carcinogênese , Colite/genética , Colite/patologia , Humanos , Inflamação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Lipocalina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold,consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomol arrange against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30 pM were isolated with only a single round of ribosome display,an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications.
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Repetição de Anquirina/fisiologia , Desenho de Fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Humanos , Proteína Quinase 1 Ativada por Mitógeno/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Biblioteca de Peptídeos , Conformação Proteica , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/química , Ressonância de Plasmônio de SuperfícieRESUMO
BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family.
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Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
INTRODUCTION: Inflammatory bowel disease (IBD) is a systemic inflammatory condition that affects the entire organism, not only the bowel. An impaired interaction with microbiota has been shown to be important. We looked for bacterial factors, which may contribute to the well-known higher incidence of poor reproductive outcome in IBD. METHODS: Urine specimen of patients with Crohn's disease (N=42), ulcerative colitis (N=46), and randomly selected patients attending the General Internal Medicine Outpatient Clinic of the Charité for non-IBD related medical conditions (N=49) was analyzed for bacteria adherent to desquamated epithelial cells and diffusely distributed bacteria in the urine using fluorescence in situ hybridization. RESULTS: The urine of IBD patients contained significantly more often Gardnerella vaginalis biofilms (CD 38%, UC 43%) than those of the control group (16%). There was no link between current disease activity, history of and present fistula and G. vaginalis biofilms, but the samples of patients with steroid refractory/dependent disease were significantly more often G. vaginalis biofilm positive. No significant differences in number of epithelial cells and leukocytes, and total bacterial counts were present. CONCLUSIONS: There is a significant link between IBD and G. vaginalis biofilm. This observation suggests an epithelial barrier dysfunction of the genital tract. Since G. vaginalis is believed to be one of the reasons responsible for bacterial vaginosis, it may be an important factor in the well-known higher incidence of poor reproductive outcome in IBD. Excessive G. vaginalis biofilms in steroid refractory/dependent disease suggests a need to avoid long-term steroid therapy.
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Doenças Urogenitais Femininas/etiologia , Gardnerella vaginalis , Infecções por Bactérias Gram-Positivas/etiologia , Doenças Inflamatórias Intestinais/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Biofilmes , Estudos de Casos e Controles , Colite Ulcerativa/complicações , Colite Ulcerativa/microbiologia , Doença de Crohn/complicações , Doença de Crohn/microbiologia , Feminino , Doenças Urogenitais Femininas/microbiologia , Humanos , Hibridização in Situ Fluorescente , Doenças Inflamatórias Intestinais/microbiologia , Masculino , Doenças Urogenitais Masculinas/etiologia , Doenças Urogenitais Masculinas/microbiologia , Pessoa de Meia-Idade , Vaginose Bacteriana/etiologia , Adulto JovemRESUMO
BACKGROUND: We analysed data on bacterial vaginosis (BV) contradicting the paradigm of mono-infection. METHODOLOGY: Tissues and epithelial cells of vagina, uterus, fallopian tubes and perianal region were investigated using fluorescence in situ hybridization (FISH) in women with BV and controls. RESULTS: Healthy vagina was free of biofilms. Prolific structured polymicrobial (StPM) Gardnerella-dominated biofilm characterised BV. The intact StPM-Gardnerella-biofilm enveloped desquamated vaginal/prepuce epithelial cells and was secreted with urine and sperma. The disease involved both genders and occurred in pairs. Children born to women with BV were negative. Monotherapy with metronidazole, moxifloxacin or local antiseptics suppressed but often did not eradicate StPM-Gardnerella-biofilms. There was no BV without Gardnerella, but Gardnerella was not BV. Outside of StPM-biofilm, Gardnerella was also found in a subset of children and healthy adults, but was dispersed, temporal and did not transform into StPM-Gardnerella-biofilm. CONCLUSIONS: StPM-Gardnerella-biofilm is an infectious subject. The assembly of single players to StPM-Gardnerella-biofilm is a not trivial every day process, but probably an evolutionary event with a long history of growth, propagation and selection for viability and ability to reshape the environment. The evolutionary memory is cemented in the structural differentiation of StPM-Gardnerella-biofilms and imparts them to resist previous and emerging challenges.
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Biofilmes/crescimento & desenvolvimento , Gardnerella/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Vaginose Bacteriana/microbiologia , Biofilmes/efeitos dos fármacos , Candidíase Vulvovaginal/microbiologia , Estudos de Casos e Controles , Feminino , Gardnerella/efeitos dos fármacos , Gardnerella/genética , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Hibridização in Situ Fluorescente , Masculino , Gravidez , Vagina/microbiologia , Vaginose Bacteriana/tratamento farmacológicoRESUMO
Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.
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Antineoplásicos Hormonais/farmacologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Repetição de Anquirina , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Hormonais/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Mapeamento de Epitopos , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química , Proteínas Recombinantes de Fusão/química , Células Sf9 , Transdução de Sinais , Spodoptera , TrastuzumabRESUMO
The myocardin-related transcription factor MAL/MRTF-A relates changes in G-actin to SRF-mediated gene expression. The set of G-actin-controlled SRF target genes includes components of the cytoskeleton and cell migration machinery as well as various signal transducers. Our previous screen for G-actin regulated genes identified a number of targets with well-established anti-proliferative and proapoptotic functions. Here, we report that the two proapoptotic Bcl-2 family members Bok and Noxa/Pmaip are directly transcriptionally induced by activated MAL and upon activation of the actin-MAL-SRF pathway. This activation depends on MRTFs and is sensitive to the actin drug latrunculin but insensitive to p53 depletion. A cis-regulatory element comprising a CArG-like box in the Bok promoter is required and inducibly recruits MAL and SRF. Moreover, MAL/MRTF-dependent transcriptional activity and target gene expression is upregulated upon stimulation of fibroblasts with TNF and staurosporin. This is accompanied by nuclear accumulation of MRTFs. The results suggest a role for MAL in TNF signaling and implicate the MAL-SRF transcription module in regulating the proapoptotic Bcl-2 family network.
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Apoptose , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transativadores/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Estaurosporina/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.
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Antibacterianos/administração & dosagem , Compostos Aza/administração & dosagem , Biofilmes/efeitos dos fármacos , Gardnerella vaginalis/efeitos dos fármacos , Gardnerella vaginalis/crescimento & desenvolvimento , Quinolinas/administração & dosagem , Vaginose Bacteriana/tratamento farmacológico , Adulto , Feminino , Fluoroquinolonas , Humanos , Metronidazol/uso terapêutico , Pessoa de Meia-Idade , Moxifloxacina , Fatores de Tempo , Resultado do Tratamento , Urina/microbiologia , Vagina/microbiologia , Vagina/cirurgia , Vaginose Bacteriana/microbiologia , Adulto JovemRESUMO
We tested whether the bacterial biofilm typical for bacterial vaginosis (BV) can be found on desquamated epithelial cells in cryopreserved donor semen. Bacteria were detected with FISH. Bacterial biofilm, covering the epithelial layer in vaginal biopsies of 20 women with BV, was evaluated on desquamated epithelial cells found in the urine of these same women and their male partners (N=20) and compared with the bacterial biofilm found on desquamated epithelial cells in randomly selected cryopreserved semen samples (N=20). Urine from 20 healthy women of laboratory and clinic personnel and urine from their partners were used as controls. Desquamated epithelial cells covered with a polymicrobial Gardnerella biofilm were identified in urine samples from all women with BV and 13 of their male partners and in none of the female controls and their partners. Gardnerella biofilm, typical for BV, was found in the semen of three of the 20 donors. Donor semen might be a vector for BV.